Nano-plasmonic molecular probes and methods of use

ABSTRACT

Plasmonics-active nanoprobes are provided for detection of target biomolecules including nucleic acids, proteins, and small molecules. The nucleic acids that can be detected include RNA, DNA, mRNA, microRNA, and small nucleotide polymorphisms (SNPs). The nanoproprobes can be used in vito in sensitive detection methods for diagnosis of diseases and disorders including cancer. Multiplexing can be performed using the nanoprobes such that multiple targets can be detected simultaneously in a single sample. The methods of use of the nanoprobes include detection by a visible color change. The nanoprobes can be used in vivo for treatment of undesireable cells in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/861,353 filed Sep. 22, 2015, which is acontinuation application of International Patent Application No.PCT/US2013/059312 filed Sep. 11, 2013, which claims priority to U.S.Provisional Patent Application No. 61/804,346 filed Mar. 22, 2013, thedisclosures of which are incorporated herein by reference in theirentireties. This application is related to U.S. patent application Ser.No. 14/024,565 filed Sep. 11, 2013, U.S. patent application Ser. No.13/888,226 filed May 6, 2013, and U.S. patent application Ser. No.13/971,822 filed Aug. 20, 2013, the disclosures of which areincorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with U.S. Government support under the NationalInstitutes of Health Grant No. T32 EB001040 and the Defense AdvancedResearch Projects Agency Grant No. HR0011-13-2-003. The U.S. Governmenthas certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates to nano-plasmonic molecular probes andtheir methods of use for in vitro and in vivo detection, diagnosis andtherapy.

BACKGROUND

In plasmonics and enhanced electromagnetic fields there are two mainsources of electromagnetic enhancement: (1) the laser electromagneticfield is enhanced due to the addition of a field caused by thepolarization of the metal particle; (2) in addition to the enhancementof the excitation laser field, there is another enhancement due to themolecule radiating an amplified emission (luminescence, Raman, etc.)field, which further polarizes the metal particle, thereby acting as anantenna to further amplify a Raman/Luminescence signal.

Electromagnetic enhancements are divided into two main classes: a)enhancements that occur only in the presence of a radiation field, andb) enhancements that can occur even in the absence of a radiation field.The first class of enhancements is further divided into severalprocesses. Plasma resonances on the substrate surfaces, also calledsurface plasmons, provide a major contribution to electromagneticenhancement. An effective type of plasmonics-active substrate consistsof nanostructured metal particles, protrusions, or rough surfaces ofmetallic materials. Incident light irradiating these surfaces excitesconduction electrons in the metal, and induces excitation of surfaceplasmons leading to Raman/Luminescence enhancement. At the plasmonfrequency, the metal nanoparticles (or nanostructured roughness) becomepolarized, resulting in large field-induced polarizations and thus largelocal fields on the surface. These local fields increase theLuminescence/Raman emission intensity, which is proportional to thesquare of the applied field at the molecule. As a result, the effectiveelectromagnetic field experienced by the analyte molecule on thesessurfaces is much larger than the actual applied field. This fielddecreases as 1/r³ away from the surface. Therefore, in theelectromagnetic models, the luminescence/Raman-active analyte moleculeis not required to be in contact with the metallic surface but can belocated anywhere within the range of the enhanced local field, which canpolarize this molecule. The dipole oscillating at the wavelength λ ofRaman or luminescence can, in turn, polarize the metallic nanostructuresand, if λ is in resonance with the localized surface plasmons, thenanostructures can enhance the observed emission light (Raman orluminescence).

Plasmonics-active metal nanoparticles also exhibit strongly enhancedvisible and near-infrared light absorption, several orders of magnitudemore intense compared to conventional laser phototherapy agents. The useof plasmonic nanoparticles as highly enhanced photoabsorbing agents hasthus introduced a much more selective and efficient phototherapystrategy.

One of several phenomena that can enhance the efficiency of lightemitted (Raman or luminescence) from molecules adsorbed on or near ametal nanostructure is Raman scatter known as the surface enhanced Ramanscattering (SERS) effect. The use of SERS measurement for a variety ofchemicals including several homocyclic and heterocyclic polyaromaticcompounds has been reported. [T. Vo-Dinh, M. Y. K. Hiromoto, G. M. Begunand R. L. Moody, “Surface-enhanced Raman spectroscopy for trace organicanalysis,” Anal. Chem., vol. 56, 1667, 1984]. Extensive research hasbeen devoted to understanding and modeling the Raman enhancement in SERSsince the mid 1980's. For example, Kerker published models ofelectromagnetic field enhancements for spherical silver nanoparticlesand metallic nanoshells around dielectric cores as far back as 1984 [M.M. Kerker, Acc. Chem. Res., 17, 370 (1984)]. Kerker's work illustratedtheoretical calculations of electromagnetic enhancements for isolatedspherical nanospheres and nanoshells at different excitationwavelengths. In his calculations, the intensity of the normally weakRaman scattering process was increased by factors as large as 10¹³ or10¹⁵ for compounds adsorbed onto a SERS substrate, allowing forsingle-molecule detection. As a result of the electromagnetic fieldenhancements produced near nanostructured metal surfaces, nanoparticleshave found increased use as fluorescence and Raman nanoprobes.

The theoretical models indicate that it is possible to tune the size ofthe nanoparticles and the nanoshells to the excitation wavelength.Experimental evidence suggests that the origin of the 10⁶- to 10¹⁵-foldRaman enhancement primarily arises from two mechanisms: a) anelectromagnetic “lightning rod” effect occurring near metal surfacestructures associated with large local fields caused by electromagneticresonances, often referred to as “surface plasmons”; and b) a chemicaleffect associated with direct energy transfer between the molecule andthe metal surface.

According to classical electromagnetic theory, electromagnetic fieldscan be locally amplified when light is incident on metal nanostructures.These field enhancements can be quite large (typically 10⁶- to 10⁷-fold,but up to 10¹⁵-fold enhancement at “hot spots”). When a nanostructuredmetallic surface is irradiated by an electromagnetic field (e.g., alaser beam), electrons within the conduction band begin to oscillate ata frequency equal to that of the incident light. These oscillatingelectrons, called “surface plasmons,” produce a secondary electric fieldwhich adds to the incident field. If these oscillating electrons arespatially confined, as is the case for isolated metallic nanospheres orroughened metallic surfaces (nanostructures), there is a characteristicfrequency (the plasmon frequency) at which there is a resonant responseof the collective oscillations to the incident field. This conditionyields intense localized field enhancements that can interact withmolecules on or near the metal surface. In an effect analogous to a“lightning rod,” secondary fields are typically most concentrated atpoints of high curvature on the roughened metal surface. It has beenwidely accepted that the electromagnetic (EM) enhancement contributesthe main part of enormous enhancement factor which greatly increases theintrinsically weak normal Raman scattering cross-section. Theoreticalstudies of EM effects have shown that the enhanced EM fields areconfined within only a tiny region near the surface of the particles,and the SERS enhancement (G) falls off as G=[r/(r+d)]^(1/2) for a singlemolecule located a distance d from the surface of a metal nanoparticleof radius r [K. Kneipp, H. Knepp, I. Itzkan, R. R Dasar, M. S. Feld, J.phys. Condens. Matter 14, R597 (2002)]. Thus, the EM enhancement factorG strongly decreases with increased distance between the analyte andmetal surface.

A label-free detection system that uses a SERS-based “MolecularSentinel” (MS) probe for multiplexed detection of gene targets has beenpublished [T. Vo-Dinh, “SERS Molecular Probe for Diagnostics and Therapyand Methods of Use Thereof”, U.S. Pat. No. 7,951,535 (2011)]. The MSnanoprobe is composed of a DNA hairpin probe (30-45 nucleotides) andmetal nanoparticles. One end of the hairpin is tagged with a SERS-activelabel. At the other end, the probe is modified with a thiol group tocovalently bond with the nanoparticle. The sequence within the loopregion is complementary to the specific sequence being targeted fordetection. In the absence of the target, the Raman label is in closeproximity to the metal surface (closed state), and a strong SERS signalis detected due to the ‘plasmonic’ enhancement mechanism near themetallic nanoparticle. The SERS enhancement (G) falls off asG=[r/(r+d)]^(1/2) for a single analyte molecule located a distance dfrom the surface of a metal nanoparticle of radius r. Theelectromagnetic SERS enhancement strongly decreases with increaseddistance, due to a total intensity decay of (1/d)^(1/2). In the presenceof the specific DNA target, hybridization disrupts the stem-loopconfiguration (open state) and separates the Raman label from the metalnanoparticle. The SERS signal is therefore significantly quenched.

Molecular sentinels (MS) have been used to detect single nucleotidepolymorphisms (SNPs) in a multiplex fashion. Specifically, the MSplasmonic nanoprobe method has be used to perform multiplex detection ofinvasive breast cancer markers in a homogenous solution assay withoutwashing or separation steps. This design comprised two MS nanoprobes,EBRR2-MS and KI-67-MS, to target the erbB-2 and ki-67 cancer genes,respectively. The results showed that only the SERS peaks associatedwith the complementary MS nanoprobes were significantly quenched when inthe presence of the target DNA.

In addition to the EM enhancement contributed from individual particles,it has been observed that the EM field is particularly strong in theinterstitial space between the particles. It is believed that theanomalously strong Raman signal originates from “hot spots”, i.e.,regions where clusters of several closely-spaced nanoparticles areconcentrated in a small volume. The high-intensity SERS then originatesfrom the mutual enhancement of surface plasmon local electric fields ofseveral nanoparticles that determine the dipole moment of a moleculetrapped in a gap between metal surfaces. This effect is also referred toas interparticle coupling or plasmonic coupling in a network ofnanoparticles (NPs), and the effect can produce a further enhancement inaddition to the enhancement from individual particles. The problem ofpredicting the electromagnetic field in the gaps between metalnanoparticles under optical illumination has attracted interest inrecent years because of the very large field enhancements induced in theparticle gaps arising from surface plasmon resonances.

To investigate this feature, the electric field was calculatedsurrounding a finite chain of metal nanospheres or nanospheroids whenilluminated with coherent light [S. J. Norton and T. Vo-Dinh, “Opticalresponse of linear chains of nanospheres and nanospheroids,” J. Opt.Soc. Amer. 25, 2767-2775 (2007)]. The chain structure consists ofnanoparticles aligned closely with small gaps between them. A method wasdeveloped applicable to spheres and spheroids which avoided the use oftranslational formulas at the expense of the numerical, but allowed forstraightforward evaluation of certain simple integrals. In this work,the quasi-static approximation was assumed, but the basic approach couldbe extended to the full-wave problem, in which retardation affects wereaccounted for. The approach was illustrated by computing the electricfield in the gaps between two spheres and between two spheroids over arange of frequencies so that the induced plasmon resonances wereevident. At frequencies matching the plasmon resonances, very largefield enhancements were observed to occur. It was also demonstrated howthe field enhancement varied with the aspect ratio of a prolatespheroid.

Plots were generated showing the calculated values of the magnitude ofthe electric field between two spheres and between two prolate spheroidswith two different aspect ratios. The plots showed the calculated valueof the field magnitude over a range of wavelengths at a point on axis inthe gap midway between the two particles. The magnitude of the incidentelectric field was unity; thus, the plots showed the field enhancementrelative to the incident field. The observed peaks corresponded to thefrequencies of the plasmon resonances. Because of the assumption of auniform incident electric field (the quasi-static approximation), theenhancement is scale invariant; that is, the enhancement only depends onthe ratio of the gap width to the particle size (e.g., the radius of asphere or, for a spheroid, the lengths of the semi-major and semi-minoraxes).

In the calculations, three pairs of particles were compared withdifferent gaps between them: a pair of identical spheres of unit radius,and a pair of prolate spheroids with two different aspect ratios butequal in volume to that of the sphere. It was noted that the plasmonresonance red-shifted with increasing aspect ratio. In addition, for agiven gap width, the two spheroids produced a noticeably largerenhancement than the two spheres. This was expected, since the smallercurvature at the spheroid ends creates a larger surface charge densityand a larger field. The increased field that was observed at the endswas attributed to the “lighting rod effect.” The pair of nanosphereshaving an aspect ratio of 4 and a 2% gap showed an electric fieldenhancement in the gap of over 700 at the peak of the plasmon resonance.The total SERS signal was approximately proportional to the fourth powerof the electric field magnitude, giving a total SERS enhancement of over4×10¹⁰. However, a spatially averaged enhancement would be much lessthan this observed peak value. [Ref: S. J. Norton and T. Vo-Dinh,“Optical response of linear chains of nanospheres and nanospheroids,” J.Opt. Soc. Amer. 25, 2767-2775 (2007)].

The detection of nucleic acid (DNA or RNA) sequences is critical formany applications ranging from clinical diagnostics, environmentalmonitoring, food safety inspection, to homeland security. For medicalapplications nucleic acid biomarkers, such as DNA, mRNA and microRNA,have long been considered as valuable diagnostic indicators to monitorthe presence of diseases and their progression. These biomarkers havegreat potential for early diagnosis and as therapeutic targets foreffective treatment of diseases. Therefore, much effort has been devotedto the development of sensitive, selective and practical techniques forthe detection of nucleic acid biomarkers.

There has been increasing interest in the use of surface-enhanced Ramanscattering (SERS) for detection of nucleic acid sequences of interest(e.g. nucleic acid sequences associated with a given disease). The SERSeffect greatly increases the Raman scattering cross-section enabling theuse of SERS for extremely sensitive detection of the analytes. Theenhancement mechanism for SERS mainly comes from intense localizedelectromagnetic (EM) fields arising from surface plasmon resonance inmetallic nanostructures with sizes on the order of tens of nanometers.Reports on the large SERS enhancement factors of 10¹²-10¹⁵ have inspiredthe development of new sensing materials allowing sensitive detection ofanalytes, even down to single molecules. Together with the narrowlinewidth and the molecular specific vibrational bands, SERS has nowbeen considered as a powerful spectroscopy approach for biochemicalanalysis and medical diagnostics.

With recent advances in nanotechnology, a variety of differentapproaches have been developed to detect DNA or RNA molecules usingSERS-active metallic (e.g. silver and gold) nanoparticles ornanostructured substrates. A variety of SERS plasmonic platforms havebeen developed for chemical and biological sensing, including alabel-free detection system that uses SERS-based “Molecular Sentinel”(MS) nanoprobes for multiplexed detection of gene targets. The MSnanoprobe consists of a “stem-loop” DNA probe having a Raman labelmolecule at one end and a metallic nanoparticle at the other end. Thedetection principle of MS is based on the plasmonic enhancementmechanism near the metallic nanoparticle (i.e. the enhanced EM fieldsare confined near the surface of metallic nanoparticles). Uponrecognition of targets, hybridization between stem-loop probes andtarget strands disrupts the stem-loop configuration and moves the Ramanlabel away from the metal surface. This switches the probe conformationfrom a closed stem-loop structure to an open linear duplex, leading to adecrease in the SERS signal (“On-to-Off”) as the SERS enhancementstrongly decreases with increased distance between the dye and metallicnanostructured surface.

New nano-plasmonic compositions having improved properties and methodsof use are desireable to take advantage of the tunability of thespectral properties of the metal nanoparticles.

SUMMARY OF THE INVENTION

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone metal nanoparticle; an oligonucleotide attached at one end to thenanoparticle, the oligonucleotide including a stem-L and a stem-Rsequence capable of hybridizing to form a hairpin structure and aplaceholder binding sequence in between the stem-L and stem-R sequences;a placeholder nucleic acid complementary to the placeholder bindingsequence and complementary to the target, wherein the placeholdernucleic acid is hybridized to the placeholder sequence in the absence ofthe target such that formation of the hairpin structure is prevented;and an optical label attached to the oligonucleotide, irradiating thesample with electromagnetic radiation from an excitation source; anddetecting the electromagnetic radiation originated by the label, whereina level of electromagnetic radiation originated by the label in thepresence of the target is changed due to movement of the label into thevicinity of the nanoparticle electromagnetic enhancement upon formationof the hairpin structure.

In one embodiment, a nanoprobe is provided for detecting nucleic acidtargets, comprising: at least one metal nanoparticle; an oligonucleotideattached at one end to the nanoparticle, the oligonucleotide including astem-L and a stem-R sequence capable of hybridizing to form a hairpinstructure and a placeholder binding sequence in between the stem-L andstem-R sequences; a placeholder nucleic acid complementary to theplaceholder binding sequence and complementary to the target, whereinthe placeholder nucleic acid is hybridized to the placeholder sequencein the absence of the target such that formation of the hairpinstructure is prevented; and an optical label attached to theoligonucleotide.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone silver-coated gold nanostar resulting from a process comprisingreducing aqueous silver (Ag⁺) to solid silver (Ag⁰) onto gold nanostarseeds under conditions such that the silver-coated gold nanostars areproduced; an oligonucleotide attached at one end to the nanoparticle,the oligonucleotide including a stem-L and a stem-R sequence capable ofhybridizing to form a hairpin structure and a placeholder bindingsequence in between the stem-L and stem-R sequences; a placeholdernucleic acid complementary to the placeholder binding sequence andcomplementary to the target, wherein the placeholder nucleic acid ishybridized to the placeholder sequence in the absence of the target suchthat formation of the hairpin structure is prevented; and an opticallabel attached to the oligonucleotide; irradiating the sample withelectromagnetic radiation from an excitation source; and detecting theelectromagnetic radiation originated by the label, wherein a level ofelectromagnetic radiation originated by the label in the presence of thetarget is changed due to movement of the label into the vicinity of thenanoparticle electromagnetic enhancement upon formation of the hairpinstructure.

In one embodiment, a method is provided for detecting protein and smallmolecule targets, comprising: contacting a nanoprobe directed to aprotein target or a small molecule target with the target underconditions suitable for the nanoprobe to bind to the target, wherein thenanoprobe comprises: at least one metal nanoparticle; an oligonucleotideattached at one end to the nanoparticle, the oligonucleotide including astem-L and a stem-R sequence capable of hybridizing to form a hairpinstructure and a placeholder binding sequence in between the stem-L andstem-R sequences; a placeholder aptamer bound to the placeholder bindingsequence such that formation of the hairpin structure is prevented,wherein the placeholder aptamer is capable of binding to the target; andan optical label attached to the oligonucleotide, irradiating the samplewith electromagnetic radiation from an excitation source; and detectingthe electromagnetic radiation originated by the label, wherein a levelof electromagnetic radiation originated by the label in the presence ofthe target is changed due to movement of the label into the vicinity ofthe nanoparticle electromagnetic enhancement upon formation of thehairpin structure.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a first and a second nanoprobe directedto a nucleic acid target with the target under conditions suitable forthe target to hybridize with the nanoprobes, wherein the first and thesecond nanoprobes comprise: at least one metal nanoparticle; anoligonucleotide probe attached at one end to the nanoparticle, the probeof the first nanoprobe including a sequence that is complementary to afirst half of the target and the probe of the second nanoprobe includinga sequence that is complementary to a second half of the target; and afirst label attached to the first probe and a separate second labelattached to the second probe, irradiating the sample withelectromagnetic radiation from an excitation source; and detecting theelectromagnetic radiation originated by both of the first and secondlabels, wherein a level of electromagnetic radiation originated by thelabels in the presence of the target is changed upon hybridization ofthe probes with the target due to movement of the labels in between thenanoparticles.

In one embodiment, a pair of nanoprobes are provided for detectingnucleic acid targets, each of a first and a second nanoprobe comprising:at least one metal nanoparticle; an oligonucleotide probe attached atone end to the nanoparticle, the probe of the first nanoprobe includinga sequence that is complementary to a first half of a target and theprobe of the second nanoprobe including a sequence that is complementaryto a second half of the target; and a first label attached to the firstprobe and a separate second label attached to the second probe.

In one embodiment, a method is provided for detecting protein targets,comprising: contacting a first and a second nanoprobe directed to aprotein target with the target under conditions suitable for thenanoprobes to bind to the target, wherein the first and the secondnanoprobes comprise: at least one metal nanoparticle; a bioreceptorattached to the nanoparticles, the bioreceptor of the first nanoprobecapable of binding to a first site on the protein target and thebioreceptor of the second nanoprobe capable of binding to a second siteon the protein target; and a first label attached to the firstbioreceptor and a separate second label attached to the secondbioreceptor, irradiating the sample with electromagnetic radiation froman excitation source; and detecting the electromagnetic radiationoriginated by both of the first and second labels, wherein a level ofelectromagnetic radiation originated by the labels in the presence ofthe target is changed upon binding of each of the bioreceptors to thetarget due to movement of the labels in between the nanoparticles.

In one embodiment, a pair of nanoprobes are provided for detectingprotein targets, each of a first and a second nanoprobe comprising: atleast one metal nanoparticle; a bioreceptor attached to thenanoparticle, the bioreceptor of the first nanoprobe capable of bindingto a first site on a protein target and the bioreceptor of the secondnanoprobe capable of binding to a second site on the protein target; anda first label attached to the first bioreceptor and a separate secondlabel attached to the second bioreceptor.

In one embodiment, a method is provided for detecting protein targets,comprising: contacting a nanoprobe comprising: at least one metalnanoparticle; a ligand attached to the nanoparticle capable of bindingto a protein target; and an optical label attached to the nanoparticle,with the target under conditions suitable for both the nanoprobe to bindto the target and for the nanoparticles to self assemble into closelypacked arrays in the absence of the target such that electromagneticfield enhancement occurs between neighboring nanoparticles, irradiatingthe sample with electromagnetic radiation from an excitation source; anddetecting the electromagnetic radiation originated by the label, whereina level of electromagnetic radiation originated by the label isdecreased upon binding of the ligand to the target due to movement ofthe metal nanoparticles further apart such that the label is lessaffected by electromagnetic field enhancement between neighboringnanoparticles.

In one embodiment, a nanoprobe is provided for detecting proteintargets, comprising: at least one metal nanoparticle; a ligand attachedto the nanoparticle capable of binding to a protein target; and anoptical label attached to the nanoparticle.

In one embodiment, a silver-coated gold nanostar is provided resultingfrom a process comprising reducing aqueous silver (Ag⁺) to solid silver(Ag⁰) onto gold nanostar seeds under conditions such that thesilver-coated gold nanostars are produced.

In one embodiment, a nanoprobe is provided comprising: a silver-coatedgold nanostar resulting from a process comprising reducing aqueoussilver (Ag⁺) to solid silver (Ag⁰) onto gold nanostar seeds underconditions such that the silver-coated gold nanostars are produced; andan optical label capable of absorbing electromagnetic radiationoriginated as a result of excitation of the nanostar with excitationradiation.

In one embodiment, a method is provided for treating undesirable cellscomprising: contacting an undesirable cell with the silver-coated goldnanostar resulting from a process comprising reducing aqueous silver(Ag⁺) to solid silver (Ag⁰) onto gold nanostar seeds under conditionssuch that the silver-coated gold nanostars are produced and having anoptical label; and irradiating the sample with electromagnetic radiationfrom an excitation source, wherein the optical label is capable ofabsorbing electromagnetic radiation from one or both of electromagneticradiation originated as a result of excitation of the nanostar anddirectly from the excitation radiation, and wherein the undesirablecells are damaged by one or both of thermal energy direct from theradiation and thermal energy emitted as a result of excitation of thenanostar.

In one embodiment, a method is provided for treating undesirable cellscomprising: contacting an undesirable cell with a nanoprobe of thepresent disclosure; and irradiating the sample with electromagneticradiation from an excitation source, wherein the optical label iscapable of absorbing electromagnetic radiation from one or both ofelectromagnetic radiation originated as a result of excitation of thenanoparticle and directly from the excitation radiation, and wherein theundesirable cells are damaged by one or both of thermal energy directfrom the radiation and thermal energy emitted as a result of excitationof the nanoparticle.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone metal nanoparticle; an oligonucleotide attached at one end to thenanoparticle, the oligonucleotide including a stem-L and a stem-Rsequence capable of hybridizing to form a hairpin structure and aplaceholder binding sequence in between the stem-L and stem-R sequences;and a placeholder nucleic acid complementary to the placeholder bindingsequence and complementary to the target, wherein the placeholdernucleic acid is hybridized to the placeholder sequence in the absence ofthe target such that formation of the hairpin structure is prevented;and detecting a color change in the presence of the target uponformation of the hairpin structure. The color change can be a visiblecolor change.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a pair of nanoprobes having at least onemetal nanoparticle and an oligonucleotide probe attached at one end tothe nanoparticle, the probe of the first nanoprobe including a sequencethat is complementary to a first half of a target and the probe of thesecond nanoprobe including a sequence that is complementary to a secondhalf of the target, with a target under conditions suitable for thetarget to hybridize with the nanoprobes; and detecting a color change inthe presence of the target upon hybridization of the probes with thetarget. The color change can be a visible color change.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a pair of nanoprobes having at least onemetal nanoparticle and a bioreceptor attached to the nanoparticle, thebioreceptor of the first nanoprobe capable of binding to a first site ona protein target and the bioreceptor of the second nanoprobe capable ofbinding to a second site on the protein target, with a target underconditions suitable for the target to bind to the nanoprobes; anddetecting a color change in the presence of the target upon binding ofthe bioreceptors to the target. The color change can be a visible colorchange.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe having at least one metalnanoparticle and a ligand attached to the nanoparticle capable ofbinding to a protein target, with a target under conditions suitable forthe target to bind to the nanoprobe; and detecting a color change in thepresence of the target upon binding of the ligand to the target. Thecolor change can be a visible color change.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofvarious embodiments, is better understood when read in conjunction withthe appended figures. For the purposes of illustration, there is shownin the Figures exemplary embodiments; however, the presently disclosedsubject matter is not limited to the specific methods and exemplaryembodiments disclosed.

FIG. 1 is a schematic diagram showing a DNA hairpin structure attachedto a metallic nanoparticle to form a molecular detector termed a“molecular sentinel” (MS). The MS involves an “On-to-Off”negative-contrast signaling scheme.

FIGS. 2A-2C illustrate an “Off-to-On” detection scheme based on an“inverse Molecular Sentinel” (iMS) nanoprobe according to embodiments ofthe present disclosure. A) The nanoprobe is shown having anoligonucleotide attached at one end to a nanoparticle (NP), theoligonucleotide including a spacer, and a stem-L and a stem-R sequencecapable of hybridizing to form a hairpin structure, a placeholderbinding sequence in between the stem-L and stem-R sequences, thenanoprobe also including a placeholder complementary to the placeholderbinding sequence and to the target (targeting region), and a Raman dyeattached to the oligonucleotide. B) The nanoprobe is similar to thatshown in (A) except that the spacer region is absent, a greater regionof the placeholder is hybridized to the placeholder binding sequence,and the stem-R region overlaps with the placeholder binding sequence. C)The nanoprobe is similar to that shown in (A) except that the stem-Rregion overlaps with the placeholder binding sequence.

FIGS. 3A-3B are diagrams illustrating the mechanism of the iMS accordingto FIG. 2. 3A) The complementary “capture probe” serves as a placeholderstrand by binding to the nucleic acid stem of the nanoprobe to keep theRaman label away from the nanoparticle surface in the ‘Off’ state. 38)Upon exposure to the “target” sequence, the capture probe leaves thenanoprobe based on competitive binding to the target, allowing thestem-loop to “close” and move the Raman label onto the nanoparticlesurface such that upon laser excitation, the Raman label experiences astrong plasmonic effect and generates an intense SERS signal, providingthe ‘On’ state.

FIGS. 4A-4B are diagrams of the iMS nanoprobe according to FIGS. 3A-3Billustrating that the capture probe can be tethered to the nanoparticlesuch that the capture probe is kept near the nanoparticle and can beresused.

FIGS. 5A-5C are schematic diagrams depicting the iMS nanoprobe accordingto FIGS. 3A-3B using temperature cycling. A) Capture probe is used tokeep the stem-loop in an open state. B) By increasing the sampletemperature, the capture probe is dehybridized from the iMS nanoprobe.C) With temperature decrease and in the presence of the target sequencethe capture probe hybridizes to the target allowing the stem-loop toclose, bringing the Raman label to the nanoparticle surface forgeneration of a SERS signal.

FIGS. 6A-6B are schematic diagrams illustrating the plasmonic nanoprobefor detection of protein and small molecule targets according toembodiments of the present disclosure.

FIGS. 7A-7B are schematic diagrams illustrating the plasmonic nanoprobefor detection of nucleic acid targets according to embodiments of thepresent disclosure.

FIGS. 8A-8B are schematic diagrams illustrating the plasmonic nanoprobefor detection of protein targets according to embodiments of the presentdisclosure.

FIG. 9 is a schematic diagram illustrating the plasmonic nanoprobe fordetection of protein targets according to embodiments of the presentdisclosure.

FIGS. 10A-10K are schematic diagrams showing various embodiments ofplasmonics-active nanoparticles of according to the present disclosure:A) Metal nanoparticle; B) Dielectric nanoparticle core covered withmetal nanocap; C) Spherical metal nanoshell covering dielectric spheroidcore; D) Oblate metal nanoshell covering dielectric spheroid core; E)Metal nanoparticle core covered with dielectric nanoshell; F) Metalnanoshell with protective coating layer; G) Multi layer metal nanoshellscovering dielectric spheroid core; H) Multi-nanoparticle structures; I)Metal nanocube and nanotriangle/nanoprism; J) Metal cylinder; and K)legend.

FIG. 11 is a schematic diagram showing the enhanced plasmonic couplingin crescent metal nanoparticles according to FIG. 10B.

FIG. 12 is a schematic diagram illustrating plasmonic nanoprobesprotected with an anti-biofouling layer made of NIPAM according toembodiments of the present disclosure. The star shape representscellular components and the wavy line represents target nucleic acidmolecules.

FIG. 13 is a schematic diagram showing preparation of plasmonic metalnanoparticles within a hollow silica shell according to embodiments ofthe present disclosure.

FIG. 14 is a schematic diagram showing release of drug molecules by achange in temperature or pH from plasmonic metal nanoparticles having aNIPAM shell according to embodiments of the present disclosure.

FIG. 15 is a schematic diagram showing a Raman data cube inmulti-spectral imaging of a microarray for multiplex NPCI detection tosimultaneously detect more than one target DNA in a solution accordingto embodiments of the present disclosure.

FIGS. 16A-16B are schematic diagrams showing use of the plasmonicnanoprobes as an in vive diagnostic according to embodiments of thepresent disclosure.

FIG. 17 is an SEM image of a plasmonics-active substrate comprising aclose-packed array of nanospheres onto which a thin metal shell ofsilver or gold has been deposited according to embodiments of thepresent disclosure.

FIGS. 18A-18B are schematic diagrams illustrating the iMS-on-Chip systemaccording to embodiments of the present disclosure.

FIGS. 19A-19B are schematic diagrams illustrating use of the iMSnanoprobes for detection using SERS, and for treatment by RNAinterference using siRNAs according to embodiments of the presentdisclosure.

FIGS. 20A-20B are schematic diagrams illustrating use of the iMSnanoprobes for detection using SERS, and for treatment by RNAinterference using anti-microRNAs according to embodiments of thepresent disclosure.

FIGS. 21A-21F are schematic diagrams showing various embodiments ofplasmonics-active nanoprobes for improved sensitivity of the presentdisclosure: A) Nanoprobe having two metal nanoparticles; B) Nanoprobehaving two metal nanotriangles; C) Nanoprobe having two metal nanocubes;D) Nanoprobe having three metal nanoparticles; E) Nanoprobe having sixmetal nanotriangles; and F) Nanoprobe having a dialetric nanoparticlecore covered with a metal nanocap.

FIG. 22 is a schematic diagram of an improved iMS nanoprobe designaccording to embodiments of the present disclosure.

FIGS. 23A-23C are a series of graphs showing SERS spectra of theRSAD2-iMS nanoprobes in the presence or absence of complementary DNAtargets according to embodiments of the present disclosure. A) Blank (notarget DNA present). B) In the presence of 1 μM non-complementary DNA(negative control). C) In the presence of 1 μM complementary target DNA.

FIGS. 24A-24C are spectra and TEM micrographs of the Ag0 and Ag7 SERSnanoprobes according to embodiments of the present disclosure. A)Comparison of Raman signal intensity from S30@Ag0-DTTC@SiO₂ (lowerspectrum) and S30@Ag7-DTTC@SiO₂ (upper spectrum), collected with a 100ms exposure time. The spectra have been background subtracted and offsetfor clarity. B) TEM micrograph of the Ag0 nanoprobes. Scale bars are 100nm. C) TEM micrograph of the Ag7 SERS nanoprobes. Scale bars are 100 nm.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to preferred embodimentsand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alteration and furthermodifications of the disclosure as illustrated herein, beingcontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e. at least one) of the grammatical object of the article. By wayof example, “a cell” means at least one cell and can include a number ofcells.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs.

As used herein, the term “nanostar” or “NS” means a nanoparticle whichhas a single core section with two or more protrusions emitting from thecore section of the nanoparticle. These protrusions are usually conicalor pyramidal in form, but not always.

As used herein, the terms “nanoprobe” and “nano-plasmonic probe” and“nano-plasmonic molecular probe” and “plasmonics—active nanoprobe” and“nanosensor” and “sensor” and “biosensor” are used hereininterchangeably for the purposes of the specification and claims and aremeant to refer to the molecular probes of the present disclosurecomprising one or more plasmonics-active nanoparticles and an attachedmolecular label such as, for example, a Raman dye; the molecular probesuseful for detecting biological targets including, but not limited to,nucleic acids, proteins, and cells. An inverse molecular sentinel (iMS)is one type of nanoprobe provided by the present disclosure. Thus, theiMS nanoprobe is herein referred to interchangeably as “iMS”, “iMSnanoprobe”, “iMS sensor”, “iMS biosensor”, sensor, biosensor, etc.

In one embodiment, the present disclosure provides a detection approachthat incorporates a SERS effect modulation scheme associated withmetallic nanoparticles and a DNA hairpin structure. Previously, a DNAhairpin structure was attached to a metallic nanoparticle to form amolecular detector that was termed a “molecular sentinel” (MS). The MSinvolves an “On-to-Off” negative-contrast signaling scheme shown inFIG. 1. Here, a new “Off-to-On” detection scheme is provided based on an“inverse Molecular Sentinel” (iMS) (FIG. 2). FIG. 2 illustrates the iMSnanoprobe having a Raman label at one end of an oligonucleotide that isimmobilized onto a metallic nanoparticle (NP) via a Au-thiol bond formedon the other end of the olionucleotide. The label can be attached at anydistance from the nanoparticle such that the label is not affected byelectromagnetic enhancement of the nanoparticle when the nanoprobe is inthe Off state.

FIG. 2A shows the nanoprobe having an oligonucleotide attached at oneend to a nanoparticle (NP), the oligonucleotide including a spacer, anda stem-L and a stem-R sequence capable of hybridizing to form a hairpinstructure, a placeholder binding sequence in between the stem-L andstem-R sequences, the nanoprobe also including a placeholdercomplementary to the placeholder binding sequence and to the target(targeting region), and a Raman dye attached to the oligonucleotide. Thenanoprobe in FIG. 2B is similar to that shown in FIG. 2A except that thespacer region is absent, a greater region of the placeholder ishybridized to the placeholder binding sequence, and the stem-R regionoverlaps with the placeholder binding sequence. The nanoprobe in FIG. 2Cis similar to that shown in FIG. 2C except that the stem-R regionoverlaps with the placeholder binding sequence.

FIGS. 3A and 3B illustrate the mechanism of the iMS. FIG. 3A shows thecomplementary “capture probe” serving as a placeholder strand by bindingto the nucleic acid stem of the nanoconstruct. The capture probe keepsthe Raman label away from the nanoparticle surface; the probe is “open”with low SERS signal, which is the ‘Off’ state. FIG. 3B shows that uponexposure to the “target” sequence, the capture probe leaves thenanoconstruct based on competitive binding to the target, allowing thestem-loop to “close” and move the Raman label onto the nanoparticlesurface. Upon laser excitation, the Raman label molecule experiences astrong plasmonic effect and generates an intense SERS signal, which isthe “On” or “closed” state. Because the plasmon field enhancementdecreases significantly from the surface of the NP, a molecule must belocated within a very close range (0-10 nm) of the nanostructure surfacein order to experience the enhanced local plasmon field. Afterhybridication with the target sequence the iMS can be regenerated byadding a new capture probe as a place holder.

FIGS. 4A and 4B illustrate that the capture probe can be tethered to thenanoparticle such that the capture probe is kept near the nanoparticleand can be resused.

FIGS. 5A-5C are schematic diagrams depicting the iMS biosensor usingtemperature cycling. In FIG. 5A, the capture probe is shown being usedto keep the stem-loop in an open state. FIG. 5B shows that by increasingthe sample temperature, the capture probe is dehybridized from the iMS.FIG. 5C shows that with temperature decrease and in the presence of thetarget sequence the capture probe hybridizes to the target allowing thestem-loop to close, bringing the Raman label to the nanoparticle surfacefor generation of a SERS signal.

FIGS. 6A-6B are schematic diagrams illustrating the plasmonic nanoprobefor detection of protein and small molecule targets according toembodiments of the present disclosure. The nanoprobe depicted in FIGS.6A-6B is similar to the nanoprobe shown in FIGS. 3A-3B except that thetarget can be a a small molecule as well as a protein and the captureprobe (placeholder) is an aptamer.

Various nanoparticles including silver nanospheres, gold nanospheres,nanoshells and nanostars, can be used to yield intense SERS signal of alabel at different plasmon resonance wavelengths. Looking at the iMSbiosensor of FIG. 2, the nucleic acid stem can have a thiol group at oneend for attaching to the nanoparticle and can have a Raman dye at theother end to act as a reporter. The nucleic acid stem reporter strandcan have four segments: stem-L, stem-R, spacer and placeholder. Thestem-L and stem-R segments allow the stem-loop structure to form afterthe placeholder strand binds to the target molecule and leaves thenanoconstruct. The “placeholder” or “placeholder strand” is herein usedinterchangeably with the term “capture probe”. The spacer is designed toprovide sufficient distance (over 10 nm) between the Raman dye andnanoparticle surface to reduce background SERS signal when the probe isin the open state. The placeholder (8-15 nucleotides) binds to theplaceholder strand to prevent the formation of the stem-loop structure.The placeholder strand has two segments: placeholder-C and targetingregion. The placeholder-C segment is complementary to the placeholdersegment of the stem and to the target sequences. The targeting region(20-30 nucleotides) is complementary to the target sequence.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone metal nanoparticle; an oligonucleotide attached at one end to thenanoparticle, the oligonucleotide including a stem-L and a stem-Rsequence capable of hybridizing to form a hairpin structure and aplaceholder binding sequence in between the stem-L and stem-R sequences;a placeholder nucleic acid complementary to the placeholder bindingsequence and complementary to the target, wherein the placeholdernucleic acid is hybridized to the placeholder sequence in the absence ofthe target such that formation of the hairpin structure is prevented;and an optical label attached to the oligonucleotide, irradiating thesample with electromagnetic radiation from an excitation source; anddetecting the electromagnetic radiation originated by the label, whereina level of electromagnetic radiation originated by the label in thepresence of the target is changed due to movement of the label into thevicinity of the nanoparticle electromagnetic enhancement upon formationof the hairpin structure.

In one embodiment, a nanoprobe is provided for detecting nucleic acidtargets, comprising: at least one metal nanoparticle; an oligonucleotideattached at one end to the nanoparticle, the oligonucleotide including astem-L and a stem-R sequence capable of hybridizing to form a hairpinstructure and a placeholder binding sequence in between the stem-L andstem-R sequences; a placeholder nucleic acid complementary to theplaceholder binding sequence and complementary to the target, whereinthe placeholder nucleic acid is hybridized to the placeholder sequencein the absence of the target such that formation of the hairpinstructure is prevented; and an optical label attached to theoligonucleotide.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone silver-coated gold nanostar resulting from a process comprisingreducing aqueous silver (Ag⁺) to solid silver (Ag⁰) onto gold nanostarseeds under conditions such that the silver-coated gold nanostars areproduced; an oligonucleotide attached at one end to the nanoparticle,the oligonucleotide including a stem-L and a stem-R sequence capable ofhybridizing to form a hairpin structure and a placeholder bindingsequence in between the stem-L and stem-R sequences; a placeholdernucleic acid complementary to the placeholder binding sequence andcomplementary to the target, wherein the placeholder nucleic acid ishybridized to the placeholder sequence in the absence of the target suchthat formation of the hairpin structure is prevented; and an opticallabel attached to the oligonucleotide; irradiating the sample withelectromagnetic radiation from an excitation source; and detecting theelectromagnetic radiation originated by the label, wherein a level ofelectromagnetic radiation originated by the label in the presence of thetarget is changed due to movement of the label into the vicinity of thenanoparticle electromagnetic enhancement upon formation of the hairpinstructure.

The optical label can comprises a Raman dye,3,3′-Diethylthiadicarbocyanine iodide (DTDC),3,3′-diethylthiatricarbocyanine iodide (DTTC),1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide (HITC), CY3 dye,CY3.5 dye, CY5.5 dye, CY7 dye, CY7.5 dye, a positively-chargedhydrophobic near infrared (NIR) dye, IR-780, IR-792, IR-797, IR-813,methylene blue hydrate (MB), 4-mercaptobenzoic acid (4-MBA),5,5′-dithiobis-2-nitrobenzoic acid (DTNB), 4-aminothiophenol (4ATP),fluorescein, fluorescein isothiocyanate (FITC), thionine dyes,rhodamine-based dye, crystal violet, a fluorescence label, or anabsorbance label.

Detecting the electromagnetic radiation originated by the label can beby one or more of surface enhanced Raman scattering (SERS) detection,surface-enhanced resonance Raman scattering (SERRS), fluorescencedetection and absorbance detection.

The nucleic acid target can include a DNA, an RNA, a microRNA, a mRNA,or a single polynucleotide polymorphism (SNP). The placeholder nucleicacid can include an siRNA or an anti-microRNA. The placeholder nucleicacid can be tethered to the metal nanoparticle.

The contacting of the nanoprobe with the target can occur in an ki vitroassay. The method can include increasing the temperature during thecontacting step to dehybridize the placeholder nucleic acid from theoligonucleotide.

In the method, the contacting of the nanoprobe with the target caninclude a second nanoprobe and a second target, and the second nanoprobecan include a second label that is directed to the second target. In themethod, the detecting can be performed using multiplexing such that thefirst and the second targets are detected simultaneously.

The metal nanoparticle can include silver nanoparticles, goldnanoparticles, silver nanostars, gold nanostars, silver-coated goldnanostars, bimetallic nanoparticles, multi-metallic nanoparticles,dielectric nanoparticle cores covered with metal nanoshells, ormulti-nanoparticle structures.

In the method, the contacting of the nanoprobe and the target can occurin vivo. The target nucleic acid can be a mRNA in a subject such as, forexample, a human or an animal, and the placeholder nucleic acid can besiRNA such that the subject is treated with mRNA interference therapy.

The target nucleic acid can be a microRNA in a subject such as, forexample, a human or an animal, and the placeholder nucleic acid can bean anti-microRNA such that the subject is treated with microRNAinterference therapy.

The metal nanoparticle can include a NIPAM protective coating. The metalnanoparticle can be embedded in a hollow silica shell. The targetnucleic acid can be in a subject such as, for example, a human or ananimal, and the nanoprobe can have a coating that includes a drug forrelease upon a change in temperature or pH such that the subject istreated with the drug upon the change in the temperature or the pH.

In one embodiment, a method is provided for detecting protein and smallmolecule targets, comprising: contacting a nanoprobe directed to aprotein target or a small molecule target with the target underconditions suitable for the nanoprobe to bind to the target, wherein thenanoprobe comprises: at least one metal nanoparticle; an oligonucleotideattached at one end to the nanoparticle, the oligonucleotide including astem-L and a stem-R sequence capable of hybridizing to form a hairpinstructure and a placeholder binding sequence in between the stem-L andstem-R sequences; a placeholder aptamer bound to the placeholder bindingsequence such that formation of the hairpin structure is prevented,wherein the placeholder aptamer is capable of binding to the target; andan optical label attached to the oligonucleotide, irradiating the samplewith electromagnetic radiation from an excitation source; and detectingthe electromagnetic radiation originated by the label, wherein a levelof electromagnetic radiation originated by the label in the presence ofthe target is changed due to movement of the label into the vicinity ofthe nanoparticle electromagnetic enhancement upon formation of thehairpin structure. The protein target can be present on the surface of acell such that detecting the protein results in detection of the cell.The cell can include a cancer cell.

It has been reported that the electromagnetic field (EM) is particularlystrong in the interstitial space between metal nanoparticles [Ref: Xu,H. X.; Aizpurua, J.; Kall, M.; Apell, P., Physical Review E 2000, 62(3), 4318-4324.] The anomalously strong Raman signal originates from“hot spots”, i.e., regions where clusters of several closely-spacednanoparticles are concentrated in a small volume. This effect, alsoreferred to as interparticle coupling or plasmonic coupling in a networkof NPs, can provide a further enhancement effect besides the enhancementfrom individual particles. In previous work, computation of the electricfield in the gaps between two spheres and between two spheroids over arange of frequencies also indicate the occurrence of very large fieldenhancements [Ref: Norton, S. J.; Vo-Dinh, T., Journal of the OpticalSociety of America A—Optics Image Science and Vision 2008, 25 (11),2767-2775]. While very large enhancement for a single hot spot can beachieved in such structures, the presence and location of such hotspotsis not predictable and the density of the hotspots tends to be very low.It is widely believed that SERS hot spots are created at locations wherethe EM field is strongly concentrated by the metallic nanostructures orbetween nanostructures. Creating a high density of such hot spots callsfor a systematic study in periodic nanostructures made out of metals.

To further underline the inter-particle plasmonic coupling effect, theEM field has been investigated at the hot spot between two nanoparticles(solid nanospheres or nanoshells) [T. Vo-Dinh, A. Dhawan, S. J. Norton,C. G. Khoury, H-N. Wang, V. Misra, and M. Gerhold, “PlasmonicNanoparticles and Nanowires: Design, Fabrication and Application inSensing” J. Phys. Chem. C, 114 (16), pp 7480-7488 (2010)]. Thetheoretical investigations dealt with dimers of nanoparticles andnanoshells using a semi-analytical method based on a multipole expansion(ME) and the finite-element method (FEM) in order to determine theelectromagnetic enhancement, especially at the interface areas of twoadjacent nanoparticles. Two types of dimmers are considered, onecomprised of two solid nanospheres and the other of two nanoshells.Nanoshells have been previously investigated and developed for medicalapplications. The maximum electric-field enhancement in the gap betweenthe particles occurs when the electric field of the incident light ispolarized along the dimer axis. The calculations of the electric fieldwere compared at a point in the gap midway between the two particles(solid sphere or shell) using two different numerical methods. The firstcalculation was performed using the FEM-based commercial softwarepackage COMSOL Multiphysics and the second was a semi-analyticalsolution based on a multipole expansion (ME) of the fields. In thelatter approach, the quasi-static approximation was employed, whichsignificantly simplified the ME analysis, but is known to give accurateresults when the particle size is about a tenth of a wavelength or less.Comparing the FEM results to those of the ME method demonstrated that inthis size range the quasi-static assumption is an excellentapproximation. The quasi-static approximation also has the virtue ofbeing computationally very fast as well as relatively simple to program.

Field calculations were performed using both the FEM and ME methods fortwo types of dimers: a pair of solid nanospheres and a pair ofnanoshells. In all cases, the outer diameter of the particles wasassumed to be 20 nm with a particle gap of 5 nm. In the firstcalculation, the magnitude of the electric field in the gap midwaybetween the particles was computed over a wavelength range from 300 nmto 800 nm. Three particular cases were considered: a dimer whoseparticles are nanoshells with a shell thickness of 15% and 35% of theouter shell radius, and a dimer whose particles are solid spheres. For apair of silver nanospheres with a 2% gap, the results showed an electricfield enhancement in the gap of over 700 at the peak of the plasmonresonance. In SERS measurements, the total signal is approximatelyproportional to the fourth power of the electric field magnitude, givinga total SERS enhancement of over 4×10¹⁰. The results underline the verystrong inter-particle plasmonic coupling effect [T. Vo-Dinh, A. Dhawan,S. J. Norton, C. G. Khoury, H-N. Wang, V. Misra, and M. Gerhold,“Plasmonic Nanopartides and Nanowires: Design, Fabrication andApplication in Sensing”, J. Phys. Chem. C, 114 (16), pp 7480-7488(2010)].

In one embodiment, the present disclosure provides plasmonics-activenanoprobes for nucleic acid targets. FIGS. 7A and 7B illustrate theplasmonic coupling detection concept for nucleic acid targets. FIG. 7Aillustrates two plasmonics-active metal (e.g, silver or gold)nanoparticles (NPs), each having a separate olionucleotide probesequence (which is also referred to herein as a “capture probe” or“capture probe DNA”) represented by broken lines in a lighter and darkershade of grey in FIGS. 7A-7B. The first probe DNA on the first NP has asequence identical to half of a target sequence of interest and has afirst Raman label bound at the end of the probe. The second probe DNA onthe second NP has a sequence identical to the other half of the targetsequence of interest and has a second Raman label bound at the end ofthe probe.

When the first and second NPs are mixed with the target sequence, theyhybridize to the target probe in such a way that the SERS labels are inthe middle (FIG. 7B). As a result, the two Raman labels are “trapped”between the two metal nanoparticles. Due to the interparticle plasmonicscoupling described above, upon excitation of the label molecules (e.g.,using a laser or other appropriate energy source), the electromagneticenhancement of the Raman signal is very intense, leading to extremelystrong SERS signals of the two Raman label (FIG. 7B). The increase ofthe SERS signal intensities of the two Raman labels can be used as a tomonitor and quantitatively detect the target nucleic acid sequence(e.g., DNA, RNA, microRNA, siRNA).

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a first and a second nanoprobe directedto a nucleic acid target with the target under conditions suitable forthe target to hybridize with the nanoprobes, wherein the first and thesecond nanoprobes comprise: at least one metal nanoparticle; anoligonucleotide probe attached at one end to the nanoparticle, the probeof the first nanoprobe including a sequence that is complementary to afirst half of the target and the probe of the second nanoprobe includinga sequence that is complementary to a second half of the target; and afirst label attached to the first probe and a separate second labelattached to the second probe, irradiating the sample withelectromagnetic radiation from an excitation source; and detecting theelectromagnetic radiation originated by both of the first and secondlabels, wherein a level of electromagnetic radiation originated by thelabels in the presence of the target is changed upon hybridization ofthe probes with the target due to movement of the labels in between thenanoparticles.

In one embodiment, a pair of nanoprobes are provided for detectingnucleic acid targets, each of a first and a second nanoprobe comprising:at least one metal nanoparticle; an oligonucleotide probe attached atone end to the nanoparticle, the probe of the first nanoprobe includinga sequence that is complementary to a first half of a target and theprobe of the second nanoprobe including a sequence that is complementaryto a second half of the target; and a first label attached to the firstprobe and a separate second label attached to the second probe.

In one embodiment, the present disclosure provides plasmonics-activenanoprobes for protein targets. FIGS. 8A and 8B illustrate the plasmoniccoupling detection concept for protein targets. FIG. 8A illustrates twoplasmonics-active metal (e.g, silver or gold) nanoprobes, each having adifferent attached bioreceptor. In one example, the first bioreceptorcan be an antibody that binds to a first site on a protein and thesecond bioreceptor can be an antibody that binds to a second site on thesame protein. In another example, the first and second bioreceptors canbind to a protein on the surface of a cell such as a cancer cell. Thetwo bioreceptors are represented by lighter and darker shades of grey aswell as different shapes in FIGS. 8A-8B. The first nanoprobe has a firstRaman label attached to the first bioreceptor and the second nanoprobehas a second Raman label bound to the second bioreceptor.

When the first and second nanoprobes are mixed with the target protein(or cancer cell having the target protein on the surface), thebioreceptor binding to the target brings the two Raman labels in betweenthe two nanoparticles of each of the nanoprobes (FIG. 8B). As a resultthe two Raman labels are “rapped” between the two metal nanoparticles.Due to the interparticle plasmonics coupling described herein above,upon excitation of the labels (e.g., using a laser or other appropriateenergy source), the electromagnetic enhancement of the Raman signal isvery intense, leading to extremely strong SERS signals for the two Ramanlabels (FIG. 8B). The increase of the SERS signal intensities of the twoRaman labels can be used to monitor and quantitatively detect theprotein target.

In one embodiment, a method is provided for detecting protein targets,comprising: contacting a first and a second nanoprobe directed to aprotein target with the target under conditions suitable for thenanoprobes to bind to the target, wherein the first and the secondnanoprobes comprise: at least one metal nanoparticle; a bioreceptorattached to the nanoparticles, the bioreceptor of the first nanoprobecapable of binding to a first site on the protein target and thebioreceptor of the second nanoprobe capable of binding to a second siteon the protein target; and a first label attached to the firstbioreceptor and a separate second label attached to the secondbioreceptor, irradiating the sample with electromagnetic radiation froman excitation source; and detecting the electromagnetic radiationoriginated by both of the first and second labels, wherein a level ofelectromagnetic radiation originated by the labels in the presence ofthe target is changed upon binding of each of the bioreceptors to thetarget due to movement of the labels in between the nanoparticles.

In one embodiment, a pair of nanoprobes are provided for detectingprotein targets, each of a first and a second nanoprobe comprising: atleast one metal nanoparticle; a bioreceptor attached to thenanoparticle, the bioreceptor of the first nanoprobe capable of bindingto a first site on a protein target and the bioreceptor of the secondnanoprobe capable of binding to a second site on the protein target; anda first label attached to the first bioreceptor and a separate secondlabel attached to the second bioreceptor.

FIG. 9 depicts an alternative embodiment of the nanoprobe for detectingprotein targets. As shown in FIG. 8, nanoparticles can be labeled with aSERS dye and functionalized with a ligand that binds to a protein targetof interest. The intracellular environment causes nanoparticles to selfassemble into closely packed arrays. This generates many “hot-spots” ofelectromagnetic field enhancement between neighboring particles, andthus high SERS from the dye label. Interaction of the targeting ligand(aptamer, nucleic acid, antibody) with the target increases theeffective diameter of the nanoparticle probe, spacing the metalnanoparticle cores further apart. This results in a reduction of the“hot-spots” and a decrease in the SERS signal emitted from the dyelabel. Ideally, as the SERS from the label decreases, a new SERS signalfrom the target can also be observed.

In one embodiment, a method is provided for detecting protein targets,comprising: contacting a nanoprobe comprising: at least one metalnanoparticle; a ligand attached to the nanoparticle capable of bindingto a protein target; and an optical label attached to the nanoparticle,with the target under conditions suitable for both the nanoprobe to bindto the target and for the nanoparticles to self assemble into closelypacked arrays in the absence of the target such that electromagneticfield enhancement occurs between neighboring nanoparticles, irradiatingthe sample with electromagnetic radiation from an excitation source; anddetecting the electromagnetic radiation originated by the label, whereina level of electromagnetic radiation originated by the label isdecreased upon binding of the ligand to the target due to movement ofthe metal nanoparticles further apart such that the label is lessaffected by electromagnetic field enhancement between neighboringnanoparticles.

In one embodiment, a nanoprobe is provided for detecting proteintargets, comprising: at least one metal nanoparticle; a ligand attachedto the nanoparticle capable of binding to a protein target; and anoptical label attached to the nanoparticle.

The protein target can be present on the surface of a cell such thatdetecting the protein results in detection of the cell. The cell caninclude a cancer cell.

In one embodiment, the present disclosure provides plasmonics-activenanoprobes. Plasmon resonances arise within a metallic nanoparticle fromthe collective oscillation of free electrons driven by an incidentoptical field. The plasmonic response of nanoparticles has played a rolein a growing number of applications, including surface-enhanced Ramanscattering (SERS), chemical sensing, drug delivery, photothermal cancertherapy, new photonic devices, biological analysis and medicaldiagnostics. The plasmonics-active metal nanostructures includenanoparticles and semi-nanoshells consisting of a layer of nanoparticlescoated by silver on one side (nanocaps or half-shells). Several groupshave shown that plasmon resonances of spherical shells can be tuned bycontrolling the shell thickness [M. M. Kerker, Acc. Chem. Res., 17, 370(1984); J. B. Jackson, S. L. Westcott, L. R. Hirsch, J. L. West and N.H. Halas, “Controlling the surface enhanced Raman effect via thenanoshell geometry,” Appl. Phys. Lett., vol. 82, 257-259, 2003]. Theseshells consist typically of a metallic layer over a dielectric core. Theanalysis has been extended to spheroidal shells and shows how plasmonresonances (both longitudinal and transverse modes) are influenced byboth shell thickness and aspect ratio. A number of researchers haveexamined the plasmonic response of the solid spheroidal particle intheir analysis of surface-enhanced Raman scattering, although thespheroidal shell appears not to have been investigated. Prolate andoblate spheroidal shells have been investigated and show interestingqualitative features in their plasmon resonances. Results indicate thatthe spheroidal shell presents two degrees of freedom for tuning: theshell thickness and the shell aspect ratio [S. J. Norton and T. Vo-Dinh,“Plasmonic Resonances of Nanoshells of Spheroidal Shape”, IEEE Trans.Nanotechnology, 6, 627-638 (2007)]. It has been shown thatnanostar-shaped structures can also be plasmonics-active and inducestrong SERS signals.

FIGS. 10A-10J are schematic diagrams showing the various embodiments ofplasmonics-active nanoparticles of the present disclosure: A) Metalnanoparticle; B) Dielectric nanoparticle core covered with metalnanocap; C) Spherical metal nanoshell covering dielectric spheroid core;D) Oblate metal nanoshell covering dielectric spheroid core; E) Metalnanoparticle core covered with dielectric nanoshell; F) Metal nanoshellwith protective coating layer; G) Multi layer metal nanoshells coveringdielectric spheroid core; H) Multi-nanoparticle structures; I) Metalnanocube and nanotriangle/nanoprism; and J) Metal cylinder.

FIG. 11 shows an embodiment where the plasmonics nanoparticles have a“crescent structure” partially covering a dielectric core (e.g., silica,polymeric material, etc.). The side of the crescent end producesextremely strong plasmonics enhancement. Furthermore the plasmoniccoupling between these crescent-induced enhancements can produce acombined and very strong coupling effect.

The nanoprobes of the present disclosure can be prepared using eithersilver (or gold) nanoparticle colloids. Gold nanoshells can befabricated using published methods using a mechanism involvingnucleation and then successive growth of gold nanoparticles around asilica dielectric core. In addition, the nanoprobes can include use ofnanospheres spin-coated on a solid support in order to produce andcontrol the desired roughness. The nanostructured support can besubsequently covered with a layer of silver that provides the conductionelectrons required for the surface plasmon mechanisms. Among thetechniques based on solid substrates, the methods can include usingsimple nanomaterials, such as Teflon or latex nanospheres. Teflon andlatex nanospheres are commercially available in a wide variety of sizes.The shapes of these materials are very regular and their size can beselected for optimal enhancement. These materials can consist ofisolated dielectric nanospheres (30-nm diameter) coated with silverproducing systems of half-nanoshells, referred to as nanocaps. Thenanocaps can be 300-nm diameter polymer nanospheres covered by a 100-nmthick silver nanocap (half-nanoshell) coating. The nanoparticles can besonicated to release them from the underlying substrate. The effect ofthe sphere size and metal layer thickness upon the SERS effect can beeasily investigated. By rotating the platform supporting thenanospheres, one can extend the solver coverage and produce the“crescent structures” shown in FIG. 11. The silver coated nanosphereswere found to be among the most plasmonics-active investigated. Gold canalso be used instead of silver to coat over nanoparticle materials.

In one embodiment, a silver-coated gold nanostar is provided resultingfrom a process comprising reducing aqueous silver (Ag⁺) to solid silver(Ag⁰) onto gold nanostar seeds under conditions such that thesilver-coated gold nanostars are produced.

In one embodiment, a nanoprobe is provided comprising: a silver-coatedgold nanostar resulting from a process comprising reducing aqueoussilver (Ag⁺) to solid silver (Ag⁰) onto gold nanostar seeds underconditions such that the silver-coated gold nanostars are produced; andan optical label capable of absorbing electromagnetic radiationoriginated as a result of excitation of the nanostar with excitationradiation.

Known methods can be employed to immobilize the bioreceptors to metalnanoparticles to prepare the nanoprobes of the present disclosure. By“bioreceptor” is meant the nucleic acid stem of the iMS nanoprobes ofthe present disclosure as well as the “bioreceptor” on the nanoprobesfor detecting proteins shown in FIGS. 8 and 9 that can be amino acidbased. The immobilization of biomolecules (such as, e.g., DNA, RNA, LNA,proteins, antibodies, etc.) to a solid support can use a wide variety ofmethods published in the literature. Binding can be performed throughcovalent bonds usually takes advantage of reactive groups such as amine(—NH₂) or sulfide (—SH) that naturally are present or can beincorporated into the biomolecule structure. Amines can react withcarboxylic acid or ester moieties in high yield to form stable amidebonds. Thiols can participate in maleimide coupling, yielding stabledialkylsulfides.

A solid support of interest is gold (or silver) nanoparticles. Themajority of immobilization schemes involving Au (Ag) surfaces utilize aprior derivatization of the surface with akylthiols, forming stablelinkages. Alkylthiols readily form self-assembled monolayers (SAM) ontosilver surfaces in micromolar concentrations. The terminus of theakylthiol chain can be used to bind biomolecules, or can be easilymodified to do so. The length of the akyithiol chain has been found tobe an important parameter, keeping the biomolecules away from thesurface. Furthermore, to avoid direct, non-specific DNA adsorption ontothe surface, alkylthiols can be used to block further access to thesurface, allowing only covalent immobilization through the linker[Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-7;Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-20].

Silver surfaces have been found to exhibit controlled self-assemblykinetics when exposed to dilute ethanolic solutions of alkylthiols. Thetilt angle formed between the surface and the hydrocarbon tail rangesfrom 0 to 15⁰. There is also a larger thiol packing density on silver,when compared to gold [Burges, J. D.; Hawkridge, F. M. Langmuir 1997,13, 3781-6]. After SAM formation on gold/silver nanoparticles,akyfthiols can be covalently coupled to biomolecules. The majority ofsynthetic techniques for the covalent immobilization of biomoleculesutilize free amine groups of a polypeptide (enzymes, antibodies,antigens, etc) or of amino-labeled DNA strands, to react with acarboxylic acid moiety forming amide bonds. As a general rule, a moreactive intermediate (labile ester) is first formed with the carboxylicacid moiety and in a later stage reacted with the free amine, increasingthe coupling yield. Successful coupling procedures include:

Binding Procedure Using N-Hydroxysuccinimide (NHS) and its Derivatives.

The coupling approach involves the esterification under mild conditionsof a carboxylic acid with a labile group, an N-hydroxysuccinimide (NHS)derivative, and further reaction with free amine groups in a polypeptide(enzymes, antibodies, antigens, etc) or amine-labeled DNA, producing astable amide [Boncheva, M.; Scheibler, L.; Lincoln, P.; Vogel, H.;Akerman, B. Langmuir 1999, 15, 4317-20]. NHS reacts almost exclusivelywith primary amine groups. Covalent immobilization can be achieved in aslittle as 30 minutes. Since H₂O competes with —NH₂ in reactionsinvolving these very labile esters, it is important to consider thehydrolysis kinetics of the available esters used in this type ofcoupling. The derivative of NHSO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate,increase the coupling yield by utilizing a leaving group that isconverted to urea during the carboxylic acid activation, hence favorablyincreasing the negative enthalpy of the reaction.

Binding Procedure Using Maleimide.

Maleimide can be used to immobilize biomolecules through available —SHmoieties. Coupling schemes with maleimide have been proven useful forthe site-specific immobilization of antibodies, Fab fragments, peptides,and SH-modified DNA strands. Sample preparation for the maleimidecoupling of a protein involves the simple reduction of disulfide bondsbetween two cysteine residues with a mild reducing agent, such asdithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphinehydrochloride. However, disulfide reduction will usually lead to theprotein losing its natural conformation, and might impair enzymaticactivity or antibody recognition. The modification of primary aminegroups with 2-iminothiolane hydrochloride (Traut's reagent) to introducesulfydryl groups is an alternative for biomolecules lacking them. Freesulfhydryls are immobilized to the maleimide surface by an additionreaction to unsaturated carbon-carbon bonds [Jordan, C. E., et al.,1997].

Binding Procedure Using Carbodiimide.

Surfaces modified with mercaptoaikyldiols can be activated with1,1′-carbonyldiimidazole (CDI) to form a carbonylimidazole intermediate.A biomolecule with an available amine group displaces the imidazole toform a carbamate linkage to the akylthiol tethered to the surface[Potyrailo, R. A., et al., 1998].

In one embodiment, the present disclosure provides plasmonicnanoparticle biosensors with anti-biofouling properties. Biofouling isone of the most critical factors to consider for in vive nanosensors.Therefore, for in vive use of the nanosensors of the present disclosure,an anti-biofouling layer can be designed to protect nanoparticle-basednanosensors. Poly(ethylene glycol) (PEG) coating has been used toprotect a wide variety of nanoprobes. One advantageof PEG is that PEGcan prevent formation of the hair-pin loop structure. However, becausethe PEG layer is also degraded over time, it is not a good choice forlong-term (e.g., up to 1 month) anti-biofouling.

Therefore, another ‘porous’ biomaterial is herein provided foranti-biofouling protection of the nanosensors of the present disclosure.The porous biomaterial provided can be N-Isopropylacrylamide (NIPAM),which can act as an anti-biofouling layer but also allows smallmolecules to diffuse in to react with the nanosensors (FIG. 12). TheNIPAM shell acts as a molecular sieve that can block the large cellularcompounds (e.g. blood cells, albumin, etc.) but allow for the diffusionof the target nucleic acid. NIPAM shells have been shown to act as amolecular sieve, excluding any molecules larger than the pore sizes ofthe membrane. Pore size can be controlled by using a copolymer such asN,N′-Methylenebisacrylamide. With such a shell, immune cells and bloodcells can be excluded from interacting with the nanosensors. Smallmolecules, such as mRNA, can diffuse through the capsule and react withthe nanosensors. Encapsulation of the biosensors of the presentdisclosure with NIPAM can provide a robust nanosensing platform for usein living organisms.

Additional anti-biofouling strategies are provided that can be used toretain nanoprobe functionality in a biological environment. One methodis to use thiol-PEG brushes to prevent protein adsorption on theparticle surface. The thiol group binds strongly to the nanoparticlesurface and can not be easily removed. Adding a small amount ofthiol-PEG to the nanostar solution is sufficient for functionalization.Variation of brush density and length can be optimized so that little tono impact on sensor functionality is observed. Another anti-biofoulingmethod involves the use of pNIPAM brushes. Functionalization can beperformed in the same manner as the thiol-PEG, but in this case it is anamine group that is attracted to the gold or silver surface. The loadingdensity and chain length of the pNIPAM brushes can be optimized in asimilar manner to the thiol-PEG. The pNIPAM brush functionalizednanoparticles can be further protected by crosslinking the brushes witha copolymer to form a pNIPAM shell. The pore size within the shell canbe controlled by varying the type of crosslinker and its amount. ThispNIPAM shell then acts as a molecular sieve, and pore size can be tuneddepending on the analyte of interest.

Silica coatings can also be used to protect the nanoprobe for sensing incomplex environments. A mesoporous silica shell with tunable pore sizecan protect the nanosensor from interference, while allowing enoughspace for the nanoprobe to still operate. The nanorattle, ornanoparticle encapsulated within a hollow silica shell, is also a viableoption to prevent biofouling. In one example of creating such astructure, gold nanostars can be coated with a spherical silver shell,and then a porous silica shell. Etching away the silver using H₂O₂results in a nanostar within a hollow silica shell (FIG. 13).

In another embodiment, the nanoprobes having a NIPAM shell as describedabove can be designed as drug carriers where a change in temperature orpH triggers drug release from the NIPAM shell (FIG. 14). The temperaturechange can be intrinsic (due to body temperature change) or can beextrinsically triggered outside the body using, for example, microwave,radio-frequency, MR signal or light.

One of the advantages of Raman/SERS is the ability for multiplexdetection. Employment of the SERS technique permits the use of manydifferent probe molecules, allowing the narrow band spectralcharacteristics of Raman-based probes to be used to advantage forsensitive, specific analysis of microarrays. Multiple probes, eachdesigned to detect a specific DNA target can be used and detectedsimultaneously using a multiplex detection system as described herein.

Raman spectroscopy can be used as a modality for detection in ultra-highthroughput microarray systems. Using a multispectral Raman imagingsystem, the entire emission spectrum of multiple wavelengths (˜10-100)can be collected on the entire image in the field of view. The resultingmultispectral image can be presented as a 3-D data cube as shown in FIG.15, consisting of two spatial dimensions (x, y) defining the image areaof interest as well as wavelength (λ), as the third dimension indicatedin the FIG. 15 on the Z axis.

Multiplex capability, which allows for monitoring of a large number ofmolecular processes simultaneously, is an important feature in systemsbiology research. A wide variety of luminescence labels (e.g.,fluorescent labels, chemiluminescent labels, quantum dots, etc.) havepreviously been developed for bioassays. Although detectionsensitivities achieved by luminescence techniques are adequate, thespectral overlap of the relatively large bandwidth of fluorescencespectra limit the number of labels that can be used simultaneously.Therefore, alternative techniques with improved multiplex capability areneeded. Due to the narrow bandwidths of Raman bands, the multiplexcapability of the SERS probes of the present disclosure is excellent incomparison to the other spectroscopic alternatives.

For comparison purposes, consider the detection of crystal fast violet(CFV) dye in fluorescence and SERS. The spectral bandwidth of CFV labelin the fluorescence spectrum is relatively broad (approximately 50-60 nmhalfwidth), whereas the bandwidth of the SERS spectrum of the same CFVlabel is much narrower (<0.5 nm or 3 cm⁻¹ halfwidth) (data note shown).In another example of the SERS advantage in “label multiplexing”, HIVand Hepatitis C (HCV) gene sequences were simultaneously detected byacquiring SERS spectra of a mixture of a CFV-labeled HIV gene sequenceand a BCB-labeled HCV gene sequence (data not shown). These resultsdemonstrate the advantage of SERS as a practical tool for theidentification and differentiation of multiple genes or gene expressionin diagnostics or HTS applications.

The data also indicate that use of SERS can increase the multiplexingcapability over currently used luminescence techniques by a factor ofseveral orders of magnitude. In a typical Raman spectrum, a spectralinterval of 3000 cm⁻¹ can provide 3000/3 or 10³ available individualspectral “intervals” at any given time. Even when allowing a deductionfactor of 10 due to possible spectral overlaps, it should be possible tofind 100 labels that can be used for labeling multiple probessimultaneously. This multiplex advantage is particularly useful inultra-high-throughput analyses where multiple gene targets can bescreened in a highly parallel multiplex modality. For example, a 10,000(10⁴) microarray can be labeled-multiplexed with 10-100 labels toprovide a 3-D data cube of 10⁵-10⁶ (one million) data.

A multiplex NPCI system primarily consists of an AOTF, an excitationlaser, a long pass filter, and a detector. The basic system can be usedto acquire images of samples at different wavelengths. To perform highthroughput measurements, the RMS system can be coupled to an imagingoptical system. A personal computer can be used to control a CCD, scanthe RF signal applied to the AOTF, and perform data acquisition. Thelight emitted from the microarray platform can be collected by animaging system, filtered by the AOTF, and then imaged onto a CCD. Bychanging the wavelength of the AOTF, a spectrum can be acquired as aseries of images (one for each wavelength).

A TeO₂ AOTF purchased from Brimrose Corporation, Baltimore, Md. (modelTEAF 10-45-70-S) can be used. According to the manufacturer, the AOTFhas an effective wavelength range of 450 nm to 700 nm (correspondingdrive frequency 178-100 MHz). For visible wavelengths in a telluriumoxide crystal, the applied acoustic wave is RF and can be switched veryquickly compared to other technologies. Unlike a liquid crystal tunablefilter where the bandwidth is fixed by the design and construction, anAOTF can vary the bandwidth by using closely spaced RF simultaneously.The spectral resolution given by the manufacturer for the AOTF used inthis study was 4 nm at 633 nm. The diffraction efficiency is relativelyhigh, typically about 70% at 600 nm. The optical aperture is 10 by 10 mmand the acceptance angle is greater than 30°. The drive power range was1.0 to 1.5 W. The RF generator used (Brimrose-model AT) could apply 0 to10 W of RF power and is controlled by a DOS-based computer using a16-bit computer controller board supplied by Brimrose.

The 2-D detection system uses an intensified CCD (Andor or RoperScientific). The interface to the PC-compatible computer is accomplishedvia an RS232 system. The excitation source is a HeNe or a suitable diodelaser. A CCD image of the emitted SERS is acquired and can serve as amap of gene expression (concentrations and distribution). By varying thebandpass of the AOTF, images can be acquired rapidly at selectedwavelengths, enabling different gene expression to be screened. The 2-Ddetector is oriented 5.5° from the optical axis of the AOTF (due to thediffraction angle of the AOTF at a central wavelength: 550 nm). The AOTFis placed 30-cm from the 2D-detectorto allow the separation of both thediffracted and reflection images and an iris will be placed in theoptical path to block to undiffracted light. A long-pass filter can beplaced flush against the iris to reject any remaining laser light thatmay have been scattered in the process of illumination. A glass lens (5cm diameter) is used to collect the light and form an image on the CCD.The output end of the microarray platform is placed in the object planeof the lens. The lens systems is chosen to give a total magnification tomatch the 2-D detector.

Another advantage of the nanoprobe biosensors of the present disclosureis that they can be used for rapid in vitro diagnostics. For example,the color of a solution containing a nanoprobe biosensor of the presentdisclosure can change rapidly and visibly in the presense of the targetof interest. As a result, the nanoprobes of the present disclosure canbe used for rapid, simple and inexpensive detection. Such a test isappropriate for environmental sensing (e.g., detecting E coli in wastestreams) and global health (e.g., detecting infectious diseases) inunderserved regions where access to sophisticated diagnostics facilitiesare not possible.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe directed to a nucleic acidtarget with the target under conditions suitable for the target tohybridize with the nanoprobe, wherein the nanoprobe comprises: at leastone metal nanoparticle; an oligonucleotide attached at one end to thenanoparticle, the oligonucleotide including a stem-L and a stem-Rsequence capable of hybridizing to form a hairpin structure and aplaceholder binding sequence in between the stem-L and stem-R sequences;and a placeholder nucleic acid complementary to the placeholder bindingsequence and complementary to the target, wherein the placeholdernucleic acid is hybridized to the placeholder sequence in the absence ofthe target such that formation of the hairpin structure is prevented;and detecting a color change in the presence of the target uponformation of the hairpin structure. The color change can be a visiblecolor change.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a pair of nanoprobes having at least onemetal nanoparticle and an oligonucleotide probe attached at one end tothe nanoparticle, the probe of the first nanoprobe including a sequencethat is complementary to a first half of a target and the probe of thesecond nanoprobe including a sequence that is complementary to a secondhalf of the target, with a target under conditions suitable for thetarget to hybridize with the nanoprobes; and detecting a color change inthe presence of the target upon hybridization of the probes with thetarget. The color change can be a visible color change.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a pair of nanoprobes having at least onemetal nanoparticle and a bioreceptor attached to the nanoparticle, thebioreceptor of the first nanoprobe capable of binding to a first site ona protein target and the bioreceptor of the second nanoprobe capable ofbinding to a second site on the protein target, with a target underconditions suitable for the target to bind to the nanoprobes; anddetecting a color change in the presence of the target upon binding ofthe bioreceptors to the target. The color change can be a visible colorchange.

In one embodiment, a method is provided for detecting nucleic acidtargets, comprising: contacting a nanoprobe having at least one metalnanoparticle and a ligand attached to the nanoparticle capable ofbinding to a protein target, with a target under conditions suitable forthe target to bind to the nanoprobe; and detecting a color change in thepresence of the target upon binding of the ligand to the target. Thecolor change can be a visible color change.

The nanosensors of the present disclosure can be used for in vivodiagnostics. FIGS. 16A-168 illustrate the use of the nanoprobes as an invivo diagnostic. The nanoprobes can be used in this manner as a realtime, permanent and continuous ‘health monitor’. For example, thenanoprobes can be given to a person by injection using variousmethodologies including: 1) deposition under the skin to form a ‘smartmole’ that can monitor a target in tissue or in the blood stream (FIG.16A); 2) nanoprobes having magnetic cores can be moved to andconcentrated in an area suitable for detection; and 3) the nanoprobescan be attached to a biocompatible material inside the skin layer.

In one example the nanoprobes can be injected as a colloidal solution inwhich the nanoprobes are polymer-coated. The nanoprobes can be embeddedinto a NIPAM hydrogel implant. The implant can be placed immediatelyunder the skin to allow for optical detection in situ. The porosity ofthe hydrogel allows for passage of the target, while excluding largerinterfering molecules. In another example, the nanoparticles of thenanoprobes can be iron oxide-gold/silver core-shell particles and thenanoprobes can be embedded in a NIPAM shell. The superparamagneic ironoxide core can be used to concentrate the nanoprobes at a specificlocation in the body with a wearable magnet. Concentration of thenanoprobes at the skin surface allows for optical interrogation throughthe skin.

In one embodiment, the nanosensors can be used to detect the hostresponse to various pathogens. Both the pathogenic nucleic acids as wellas the host response can be detected. The detection can be performed inthe cytosol of dermal cells. Nanosensors can be designed for detectionof the human radical S-adenosyl methionine domain containing 2 (RSAD2)gene, which is involved in antiviral defense and is one of the mosthighly induced genes upon interferon stimulation or infection withvarious viruses, including human cytomegalovirus (HCMV), influenzavirus, hepatilis C virus (HCV), dengue virus, alphaviruses, andretroviuses such as human immunodeficiency virus (HIV). The RSAD2 genehas emerged as a host-response biomarker for diagnosis of respiratoryinfections. In addition, nanosensors of the present disclosure can beprepared to detect critical pathogen biomarkers such as rlbE, fliC andmobA genes for Escherichia coli (E. coli) serotype O157:H7; mecA andfemA genes for Staphylococcus aureus and Staphylococcus epidermidis;aroQ and 16S rRNA genes for Erwinia herbicola; protective antigen (PA)and anthrax toxin activator (atxA) genes for Bacillus anthracis.

Several diagnostics systems can be utilized, depending on the degree ofminiaturization. For example, detection of the target can be performedusing a portable Raman diagnostic system having excitation light sourceand an optical detector (FIG. 16B). An alternative diagnostic system caninclude a pocket-sized (or palm-sized) battery-operated Ramandiagnostics system that is linked to the ‘smart mole’ by fiberopticsexcitation and detection (FIG. 168). The pocket-sized system can beoperated remotely by an iPhone or similar device. Furtherminiaturization can shrink the size of the portable diagnostic systeminto the size of a ‘wristwatch-sized’ battery-operated Raman diagnosticsdevice (FIG. 16B)

The nanoprobes and methods of use of the present disclosure are usefulfor a wide variety of applications based on DNA/RNA/protein detectionincluding, but not limited to: biomedical applications, point-of-carediagnostics, food safety, environmental monitoring, industrial processsensing, quality control applications, biotechnology industrial control,quality control, global health, cancer research, heart diseasediagnostics, homeland defense.

In addition to Raman and SERS, other photonic techniques can be used forexcitation of the nanoprobes in the methods of the present disclosure.For example, other parts of the electromagnetic spectrum can be used forexcitation, ranging from gamma rays and X rays throughout ultraviolet,visible, infrared, microwave and radio frequency energy.

The nanoprobes of the present disclosure can be integrated with biochiptechnology for proteomics and genomics applications. Rapid, simple,cost-effective medical devices for screening multiple medical diseasesand infectious pathogens can be essential for early diagnosis andimproved treatments of many illnesses. An important factor in medicaldiagnostics is rapid, selective, and sensitive detection of biochemicalsubstances (such as, e.g., nucleic acids, proteins, metabolites, andnucleic acids), biological species or living systems (bacteria, virus orrelated components) at ultratrace levels in biological samples (such as,e.g., tissues, blood and other bodily fluids) or in vive in humans andanimals using anti-biofouling schemes.

For example, the nanoprobes can be formed onto the surface of aplasmonics-active chip substrate. An example of chip substrate is aclose-packed array of nanospheres onto which a thin metal shell ofsilver or gold has been deposited (FIG. 17). This chip substrate is aninexpensive, reproducible and effective plasmonics-active substrate thatcan be used for SERS studies requiring high detection sensitivity [T.Vo-Dinh, M. Y. K. Hiromoto, G. M. Begun, and R. L. Moody,“Surface-Enhanced Raman Spectroscopy for Trace Organic Analysis,” Anal.Chem., 56: 1667 (1984); C. Khoury and T. Vo-Dinh, “Nanowave” Substratesfor SERS: Fabrication and Numerical Analysis”, J. Phys. Chem C, 116,7534-7545 (2012)].

FIGS. 18A-188 are schematic diagrams illustrating the iMS-on-Chipsystem. In this case, the iMS hairpin nanoprobes are immobilized on aplasmonic-active substrate. The SERS signal is off as the iMS probes arein the “open” or “off” state in the presense of the capture probe (FIG.18A). In the presence of target molecules, the capture probes aredisplaced by competitive hybridization and the SERS signal is turned onas the iMS probes are in the “closed” state (FIG. 188).

The nanoprobes can be integrated onto a multi-functional biochip basedon an integrated circuit photodiode array for use in medical diagnosticsand pathogen detection. The biochip can be a self-contained device whichallows simultaneous detection of various types of biotargets usingdifferent bioreceptors (e.g., antibodies, nucleic acids, enzymes,cellular probes) on a single system. The biochip sensor array device,which can be based on an integrated circuit (IC), can be designed usingcomplementary metal oxide silicon (CMOS) technology and includesphotosensors, amplifiers, discriminators and logic circuitry on board.The highly integrated biochip can be produced using the capability offabricating multiple optical sensing elements and microelectronics forup to 100 sensing channels on a single IC. The capability of large-scaleproduction using low-cost IC technology is an important advantage. Theassembly process of various components is made simple by cost-effectiveintegration of multiple elements on a single chip. The nano-plasmonicbiochip can me miniaturized such that it can be implanted into the skinand deep in tissue for real time or near real-time in vivo detection.

The nanoprobes of the present disclosure can also be used as amultifunctional nano-device for detection, imaging and therapy(theranostics). FIGS. 19 and 20 are schematic diagrams illustrating useof the iMS nanoprobes for detection using SERS, and for treatment by RNAinterference using siRNAs (FIGS. 19A-19B) and anti-microRNAs (FIGS.20A-20B). In this approach, the placeholder is a siRNA or ananti-microRNA for a specific mRNA or microRNA of interest as thetherapeutic target.

The anti-biofouling strategies described herein above for retainingnanoprobe functionality in biological environments can also be employedwith the theranostic nanoprobes. In addition, the theranostic nanoprobescan be encapsulated into nanocarriers. The family of nanocarriers can bebroadly categorized as polymer, lipid, apoferritins, surfactant andnanomaterial-based systems. Compared to micrometer and sub-micrometersize carriers such as liposomes, nanocarriers provide higher surfacearea-to-volume ratio, and have the potential to increase solubility,enhance bioavailability, improve controlled release and enable precisiontargeting of the entrapped compounds to a greater extent. As aconsequence of their improved stability and targeting, the amount oftherapeutic molecules required to exert a specific effect whenencapsulated in nanocarriers can be much less than the amount requiredwhen unencapsulated.

The plasmonics-active nanoprobes of the present disclosure can bedesigned for improved sensitivity. FIGS. 21A-20F are schematic diagramsshowing various embodiments of plasmonics-active nanoprobes for improvedsensitivity. For example, the nucleic acid stem of the iMS nanoprobe orthe bioreceptor of the nanoprobe for detection of proteins can beattached to more than one nanoparticle. This is illustrated in FIGS.21A-21F: A) Nanoprobe having two metal nanoparticles; B) Nanoprobehaving two metal nanotriangles; C) Nanoprobe having two metal nanocubes;D) Nanoprobe having three metal nanoparticles; and E) Nanoprobe havingsix metal nanotriangles. In another example, the nanoparticle of thenanoprobe can be a dialetric nanoparticle core covered with a metalnanocap F).

In one embodiment, a method is provided for treating undesirable cellscomprising: contacting an undesirable cell with the silver-coated goldnanostar resulting from a process comprising reducing aqueous silver(Ag⁺) to solid silver (Ag⁰) onto gold nanostar seeds under conditionssuch that the silver-coated gold nanostars are produced and having anoptical label; and irradiating the sample with electromagnetic radiationfrom an excitation source, wherein the optical label is capable ofabsorbing electromagnetic radiation from one or both of electromagneticradiation originated as a result of excitation of the nanostar anddirectly from the excitation radiation, and wherein the undesirablecells are damaged by one or both of thermal energy direct from theradiation and thermal energy emitted as a result of excitation of thenanostar.

In one embodiment, a method is provided for treating undesirable cellscomprising: contacting an undesirable cell with a nanoprobe of thepresent disclosure; and irradiating the sample with electromagneticradiation from an excitation source, wherein the optical label iscapable of absorbing electromagnetic radiation from one or both ofelectromagnetic radiation originated as a result of excitation of thenanoparticle and directly from the excitation radiation, and wherein theundesirable cells are damaged by one or both of thermal energy directfrom the radiation and thermal energy emitted as a result of excitationof the nanoparticle.

In the method for treating undesirable cells, the optical label caninclude a Raman dye, 3,3′-Diethylthiadicarbocyanine iodide (DTDC),3,3′-diethylthiatricarbocyanine iodide (DTTC),1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide (HITC), CY3 dye,CY3.5 dye, CY5.5 dye, CY7 dye, CY7.5 dye, a positively-chargedhydrophobic near infrared (NIR) dye, IR-780, IR-792, IR-797, IR-813,methylene blue hydrate (MB), 4-mercaptobenzoic acid (4-MBA),5,5′-dithiobis-2-nitrobenzoic acid (DTNB), 4-aminothiophenol (4ATP),fluorescein, fluorescein isothiocyanate (FITC), thionine dyes,rhodamine-based dye, crystal violet, a fluorescence label, or absorbancelabel. The method can further include detecting the electromagneticradiation originated by the optical label by one or more of surfaceenhanced Raman scattering (SERS) detection, surface-enhanced resonanceRaman scattering (SERRS), fluorescence detection, and absorbancedetection.

In the method for treating undesirable cells, detecting theelectromagnetic radiation originated by the optical label can serve tolocate the targeted undesirable cells such that the irradiating can bebetter localized to the undesirable cells. The nanoparticle can includea protective layer surrounding the nanoparticle having within the layerone or more of a photosensitizer, a photoactivator, and a chemotherapydrug such that the photosensitizer, the photoactivator, and thechemotherapy drug is released or activated via one or more of passivediffusion release, photochemically triggered release, thermal triggeredrelease, pH triggered release, photochemical activation, and thermalactivation. The protective layer can include NIPAM. The undesireablecells can include cancer cells.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1 Preparation of Sliver (Gold) Nanoparticles

Silver (or gold) colloids were prepared according to the standardLee-Meisel method: 200 mL of 10⁻³ M AgNO₃ aqueous solution was boiledunder vigorous stirring, then 5 mL of 35-mM sodium citrate solution wereadded and the resulting mixture was kept boiling for 1 h. This procedurewas reported to yield ˜10¹¹ particles/mL of homogenously sized colloidalparticles with a diameter of 35-50 nm and an absorption maximum at 390nm. The colloidal solutions were stored at 4° C. and protected from roomlight. Further dilutions of the colloidal solutions were carried outusing distilled water.

Example 2 Fabrication/Preparation of Metal Nanocaps

The approach used involved the use of nanospheres spin-coated on a solidsupport in order to produce and control the desired roughness. Thenanostructured support was subsequently covered with a layer of silverthat provides the conduction electrons required for the surface plasmonmechanisms. Among the techniques based on solid substrates, the methodsusing simple nanomaterials, such as Teflon or latex nanospheres, appearto be the simplest to prepare. Teflon and latex nanospheres arecommercially available in a wide variety of sizes. The shapes of thesematerials are very regular and their size can be selected for optimalenhancement. These materials consist of isolated dielectric nanospheres(30-nm diameter) coated with silver producing systems of hal-nanoshells,referred to as nanocaps. The nanoparticles can be sonicated to releasethem from the underlying substrate. The effect of the sphere size andmetal layer thickness upon the SERS effect can be easily investigated.By rotating the platform supporting the nanospheres, one can extend thesolver coverage and produce the “crescent structures” shown in FIG. 11.The silver coated nanospheres were found to be among the mostplasmonics-active investigated. Gold can also be used instead of silverto coat over a nanoparticles materials.

Example 3 Fabrication of Gold Nanoshells

Gold nanoshells were prepared using the method described by Hirsch etal. [Hirsch L R, Stafford R J, Bankson J A, Sershen S R, Price R E,Hazie J D, Halas N J, West J L (2003) Nanoshell-mediated near infraredthermal therapy of tumors under MR Guidance. Proc Natl Acad Sci100:13549-13554]. A mechanism was used involving nucleation and thensuccessive growth of gold nanoparticles around a silica dielectric core.Gold nanoparticles, the seed, prepared as described above using theFrens method, were used to grow the gold shell. Silica nanoparticles(100 nm) used for the core of the nanoshells were monodispersed insolution of 1% APTES in EtOH. The gold “seed” colloid synthesized usingthe Frens method were grown onto the surface of silica nanoparticles viamolecular linkage of amine groups. The “seed” covers the aminated silicananoparticle surface, first as a discontinuous gold metal layergradually growing forming a continuous gold shell. Gold nanoparticlesused as the “seed” were characterized using optical transmissionspectroscopy (UV-Vis Spectrophotometer, Beckman Coulter, Fullerton,Calif.) and atomic force microscopy (Atomic Force Microscope, VeecoInstruments, Woodbury, N.Y.) while gold nanoshells were characterizedusing optical transmission spectroscopy and scanning electron microscopy(Scanning Electron Microscope, Hitachi S-4700, Hitachi High TechnologiesAmerica, Inc. Pleasanton, N.Y.).

Example 4 SERS-Based “Off-to-On” Inverse Molecular Sentinel (IMS)Plasmonic Nanoprobes

The SERS-based “off-to-on” iMS nanoprobes are based on DNAstrand-displacement reaction and hybridization with target (see FIGS.2-4). The iMS nanoprobe is composed of a iMS hairpin nucleic acid stemattached to a plasmonic-active metallic (e.g. silver or gold)nanoparticle and a placeholder or capture probe. One end of the iMS stemwas tagged with a SERS-active label as a signal reporter.

In this experiment, 1 μM complementary target or non-complementarysingle-stranded DNA (negative control) was added to the iMS-Placeholder(signal-OFF) solution and incubated at room temperature for 2 hoursfollowed by SERS measurements. In the presence of the target sequences,a strong SERS signal was detected (data not shown). In contrast, onlyweak background signal was observed both in the blank (absence ofanalytes) sample and in the presence of non-complementary sequences(negative control). This result demonstrates that the placeholder wasreleased from the iMS allowing the iMS hairpin structure to form andswitching the SERS signal “ON”.

Example 5 Improved Inverse Molecular Sentinel (IMS) Plasmonic Nanoprobes

The effect of MCH (mercaptohexanol) on the hybridization efficiency ofthe iMS was investigated (FIG. 22). The results indicated that theaddition of MCH displaced non-specific interactions between the DNA andAg (or Au) and caused the DNA to stand up from the surface (data notshown). MCH improved the hybridization efficiency of the immobilizedprobes. In addition, addition of MCH was shown to aid in producing aneffective “off” signal (data not shown).

Several designs were investigated to optimize the capability to turn“OFF” iMS SERS signal using placeholder. In this experiment,PEGylated-iMS nanoprobes were compared with and without the addition ofMCH (mercapto-hexanol). In the first case, the iMS nanoprobe wasfunctionalized with 10-μM mPEG-350. The iMS nanoprobed in 2.5 mMMgCl₂-Tris buffer exhibited the SERS signal “ON” state. The SERS signalcould not be completely turned “OFF” in the presence of 100 nMplaceholder (data not shown). In the second case, the iMS nanoprobe wasfunctionalized with 2-μM mPEG-350, and then incubated with 20 μM-MCH.The iMS nanoprobed in 5 mM MgCl₂-Tris buffer exhibited the SERS signal“ON” state. The SERS signal was completely turned “OFF” in the presenceof 100 nM placeholder (data not shown).

Example 6 Preparation of a Plasmonics-Active Chip Substrate

The nanowave substrate fabrication involved two separate steps. Thefirst step involved washing glass cover slips by incubating them innitric acid, followed by a careful rinsing process with ultrapure DIwater. This ensured removal of organic residue on the glass surface andpopulated the surface with hydroxyl groups. The cover slips were driedin a stream of nitrogen and placed on a spin coater. Silica nanospheres(100-nm diameter, Polysciences), dispersed in a ethanol:ethylene glycolsolvent (85%:15% v/v), were dropcast on the cover slip and spun at 6000rpm for 5 seconds. The nanospheres adhered to the coverslip, producing aclose-packed array of silica nanospheres. The second step entailedcoating the sphere-covered slides with a metal (gold or silver),achieved by transfer to an E-beam evaporator. To ensure uniform metalcoating the substrates were rotated above the silver source duringdeposition, which was conducted at a vacuum pressure below 5×10⁻⁶ Torr.The desired thickness of metal was evaporated onto the array ofnanospheres, producing an array of silica@metal half-nanoshells,referred to as the Nanowave.

Example 7 Inverse Molecular Sentinel (IMS) Plasmonic Nanoprobes forNucleic Acid Detection

To further extend the use of SERS for nucleic acid detection, MSnanosensors were developed with unique properties, i.e. an “Off-to-On”detection scheme referred to as the “Inverse Molecular Sentinel” (iMS)nanoprobes (FIG. 2). In this study, the iMS detection method wasimplemented using gold nanostars as the SERS-active platform. Thenanostars are surfactant-free nanoparticles with tunable plasmonresonances and multiple sharp branches making them ideal monomeric SERSplatform due to their superior SERS enhancement factor. As shown in FIG.2, the “stem-loop” DNA probe of the iMS, having a Raman label at one endof the stem, is immobilized onto a gold nanostar via a gold-thiol bondformed on the other end of the stem. A complementary DNA probe, servingas a “placeholder” strand bound to the iMS nanoprobe, keeps the Ramandye away from the nanostar surface. Because the plasmon fieldenhancement decreases significantly from the surface, a dye moleculemust be located very close to the metal surface in order to experiencethe enhanced local plasmon field. Thus, in the absence of the target,the probe is denoted “Off” with low SERS signal, which is the “open” or“off” state. Upon exposure to the target sequences, the placeholderstrand leaves the nanostar surface based on DNA strand displacement bythe complementary target strand, allowing the stem-loop to close andmoving the Raman label onto the gold surface; this yields a strong SERSsignal, and is thus denoted “On” or “closed” state.

In this study, the human radical S-adenosyl methionine domain containing2 (RSAD2) gene was used as the model system to demonstrate feasibilityof the method. This gene is well known for its critical role in hostimmune response to viral infection. For infectious disease diagnosis,this approach involves detecting the host response to various pathogensby evaluating changes in gene expression in response to infection. Forexample, upon viral infection, type I interferons (IFNs) are producedand secreted by infected cells to initiate a complex signaling cascade,leading to the induction of hundreds of genes that limit viralinfection. Among these antiviral genes, the RSAD2 gene encoding aprotein known as Viperin has been recognized as one of the most highlyinduced genes upon interferon stimulation or infection with variousviruses. Thus, it is an excellent host-response biomarker for diagnosisof viral infections.

To test the effectiveness of the designed iMS nanoprobes for RSAD2, theCy5-labeled iMS nanoprobes were first conjugated with placeholders inPBS overnight at 37° C. to ensure that the stem-loop probes are open(signal “Off”). The iMS-placeholder conjugates were then tested withtheir complementary target DNA in PBS for 1 hour at 37° C. FIG. 23 showsthe SERS spectra of the iMS nanoprobes in the presence or absence oftarget DNA sequences: A) blank (no target DNA present); B) in thepresence of 1 μM non-complementary DNA (negative control); and C) in thepresence of 1 μM complementary target DNA. In the presence of target DNA(FIG. 23C), the SERS intensity of the major Raman bands wassignificantly increased indicating that the Cy5 dyes were in closeproximity to the nanostar surface. The increased SERS signal indicatesthat hybridization between the targets and placeholder strands enabledthe stem-loop structure of the DNA probes to close, thereby moving theSERS dye Cy5 onto the nanostar surface. In this case, the SERS signal ofthe iMS nanoprobes was turned “On”. On the other hand, in the presenceof non-complementary DNA (negative control: FIG. 23B), the SERSintensity of the major Raman bands remained similar to the blank sample(FIG. 23A), indicating that the dye molecules were separated from thegold surface by the placeholder strands; that is, the iMS nanoprobesadopted an open stem-loop configuration (SERS “Off”) in the absence oftargets.

In another experiment, the iMS nanoprobes were designed as describedabove. However, in this case, the iMS-placeholder conjugates wereincubated with their complementary target DNA for only 10 minutes ratherthan 1 hour prior to measurement of the SERS spectra. Again, in thepresence of the target DNA, the SERS intensity of the major Raman bandswas significantly increased relative to that in the absence of thetarget DNA indicating that hybridization between the target andplaceholder strands enabled the stem-loop structure of the DNA probes toclose such that the Raman dye moved onto the nanosphere surface (datanot shown).

In another experiment, the iMS nanoprobes were designed as described forthe first experiment above. However, in this case, 10 nM rather than 1μM target DNA was used. Again, in the presence of the target DNA, theSERS intensity of the major Raman bands was significantly increasedrelative to that in the absence of the target DNA indicating thathybridization between the target and placeholder strands enabled thestem-loop structure of the DNA probes to close such that the Raman dyemoved onto the nanosphere surface (data not shown).

In another experiment, Raman dye-labeled and PEGlyated iMS nanoprobeswere generated using silver nanosphere cores. As described above, thenanoprobes were first conjugated with placeholders to ensure that thestem-loop probes were open (signal “Off”). The SERS spectra of theiMS-placeholder conjugates were measured in the presence or absence of 1μl target DNA sequences. In the presence of the target DNA, the SERSintensity of the major Raman bands was significantly increased relativeto that in the absence of the target DNA indicating that hybridizationbetween the target and placeholder strands enabled the stem-loopstructure of the DNA probes to close such that the Raman dye moved ontothe nanosphere surface (data not shown).

In conclusion, the utility has been demonstrated of using the“Off-to-On” iMS nanoprobes to detect nucleic acid targets using SERS andplasmonic gold nanostars. Moreover, this iMS approach does not requirelabeling targets and post-hybridization washing steps, making the assayprocedure simple and rapid. The results from this study demonstrate theutility of the iMS approach as a diagnostic tool to detect nucleic acidbiomarkers for medical applications.

EXPERIMENTAL SECTION

DNA Sequences:

The stem-loop and placeholder DNA probes used for RSAD2 detection were5′-thiol-CTCTATAAGTGGTGTAGGGATTATAGAG-Cy5-3′ (SEQ ID NO: 1) and5′-GAAAGCGACTCTATAATCCCTACACCAC-3′ (SEQ ID NO: 2), respectively. Thesynthetic RSAD2 DNA target and non-complementary sequences used for thedemonstration were5′-AAAGCTGAGGAGGTGGTGTAGGGATTATAGAGTCGCTTTCAAGATAAATT-3′ (SEQ ID NO: 3)and 5′-TCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGAC-3′ (SEQ ID NO: 4),respectively. All oligonucleotides were purchased from Integrated DNATechnologies, Inc (Coralville, Iowa).

Preparation of Gold Nanostars:

The gold nanostars were prepared as described previously. Briefly, 12 nmseed solution was first prepared using a modified Turkevich method. Goldnanostars were then grown from the seed by simultaneous addition of 100μL of 2 mM AgNO₃ and 50 μL of 0.1 M ascorbic acid to a solutioncontaining 10 mL of 0.25 mM HAuCl₄, 10 μL of 1 N HCl, and 100 μL of the12 nm gold seed solution under vigorous stirring. After 10s, thesolution turned from light red to a dark gray. The stock concentrationof nanoparticles is approximately 0.1 nM, as determined by nanoparticletracking analysis (NTA).

Synthesis of Inverse Molecular Sentinel (IMS) Nanoprobes:

The stem-loop DNA probes were first treated with 0.1 M dithiothreitol(DTT) at room temperature for 1 hr followed by desalting in NAP-5columns (GE Healthcare) to remove the disulfide protecting groups. Thedeprotected stem-loop probes (at final concentration of 0.1 μM) werethen incubated with gold nanostars in 0.25 mM MgCl₂ solution overnightat room temperature. To stabilize the nanoprobes, 1 μM ofO-[2-(3-mercaptopropionylamino)ethyl]-O′-methylpolyethylene glycol(mPEG-SH, 5000) was added to the solution for 30 min. The gold surfaceof nanostars was then passivated using 0.1 mM 6-mercapto-1-hexanol(MCH). The functionalized nanoprobes were washed with Tris-HCl buffer(10 mM, pH 8.0) containing Tween 20 (0.01%) using repeatedcentrifugation at 7,000 rpm for 10 min. The purified nanoprobes werefinally resuspended in Tris-HCl buffer (10 mM, pH 8.0).

IMS Assay Procedure:

To turn off the iMS SERS signal, the nanoprobes were incubated with 0.1mM placeholder probes in PBS buffer solution overnight at 37° C. The“Off” iMS nanoprobes were then incubated with 1 μM target ornon-complementary sequences at 37° C. for 1 hr followed by SERSmeasurements using a Renishaw InVia confocal Raman microscope equippedwith a 632.8 nm HeNe laser. The light from the laser was passed througha laser line filter, and focused into the sample solution with a 10×microscope objective. The Raman scattered light was then detected by a1024×256 pixel R.

Example 8 Synthesis of IMS Nanoprobes with Sliver-Coated Gold Nanostarsfor SERS Detection

Nanostar Synthesis (AuNS).

Two sizes of AuNS were synthesized as previously reported. A 12 nm goldseed solution was prepared by adding 15 mL of 1% trisodium citrate to100 mL of a boiling solution of 1 mM HAuCl₄. This solution was keptboiling for an additional 15 minutes, cooled to room temperature in anice bath, filtered through a 0.22 μm nitrocellulose membrane, and storedat 4° C. until use. To produce the larger AuNS, designated S30, 100 μLof the gold seed was added to a 10 mL solution of 0.25 mM HAuCl₄containing 10 μL of 1 N HCl, immediately followed by the simultaneousaddition of 50 μL 0.1 M AA and 100 μL 3 mM AgNO₃ under moderatestirring. The smaller AuNS, designated S5, were produced in the samemanner as above, but using 0.5 mM AgNO₃ in place of 3 mM AgNO₃. Aftersynthesis, 100 μL of 0.1 M CTAB was added to the AuNS solution and leftstirring for 5 minutes. The particles were then centrifuged at 2000 rcffor 20 minutes at 4° C., the supernatant discarded, and the particlesre-dispersed in 10 mL of 1 mM CTAB solution.

Silver Coating of Gold Nanostars (AuNS@Ag).

A 1 mL aliquot of the washed AuNS solution was transferred into a 1.5 mLcentrifuge tube. The sample was briefly vortexed after each subsequentchemical addition. A small volume (varied between 0 and 15 μL) of 0.1 MAgNO₃ and an equivalent volume of 0.1 M AA were added to the solution.The reduction of silver by AA was initiated by the addition of NH₄OH(same volume as above), at which point the color of the solution beganto darken. After about 5 minutes, the solution color had stabilized,indicating completion of the reaction. The various silver-coated AuNSsamples were designated according to the volume of AgNO₃ added (e.g.,S30@Ag5 for S30 AuNS coated using 5 μL of 0.1 M AgNO₃). The silvercoated AuNS were then labeled with dye by adding 1 μM finalconcentration of the desired dye (dissolved in EtOH) to the solution,allowing it to sit for 15 minutes, centrifuging at 2000 rcf for 10minutes, discarding the supernatant, and re-dispersing in water.

Synthesis of Inverse Molecular Sentinel (IMS) Nanoprobes:

The stem-loop DNA probes were first treated with 0.1 M dithiothreitol(DTT) at room temperature for 1 hr followed by desalting in NAP-5columns (GE Healthcare) to remove the disulfide protecting groups. Thedeprotected stem-loop probes (at final concentration of 0.1 μM) werethen incubated with gold nanostars in 0.25 mM MgCl₂ solution overnightat room temperature. To stabilize the nanoprobes, 1 μM ofO-[2-(3-mercaptopropionylamino)ethyl]-O′-methylpolyethylene glycol(mPEG-SH, 5000) was added to the solution for 30 min. The gold surfaceof nanostars was then passivated using 0.1 mM 6-mercapto-1-hexanol(MCH). The functionalized nanoprobes were washed with Tris-HCl buffer(10 mM, pH 8.0) containing Tween 20 (0.01%) using repeatedcentrifugation at 7,000 rpm for 10 min. The purified nanoprobes werefinally resuspended in Tris-HCl buffer (10 mM, pH 8.0).

IMS Assay Procedure:

To turn off the iMS SERS signal, the nanoprobes were incubated with 0.1mM placeholder probes in PBS buffer solution overnight at 37° C. The“Off” iMS nanoprobes were then incubated with 1 μM target ornon-complementary sequences at 37° C. for 1 hr followed by SERSmeasurements using a Renishaw InVia confocal Raman microscope equippedwith a 632.8 nm HeNe laser. The light from the laser was passed througha laser line filter, and focused into the sample solution with a 10×microscope objective. The Raman scattered light was then detected by a1024×256 pixel R.

The iMS-placeholder conjugates were incubated with their complementarytarget DNA (1 μM) and SERS spectra were measured. As described above inExample 6, in the presence of the target DNA, the SERS intensity of themajor Raman bands was significantly increased relative to that in theabsence of the target DNA indicating that hybridization between thetarget and placeholder strands enabled the stem-loop structure of theDNA probes to close such that the Raman dye moved onto the nanospheresurface (data not shown).

In a similar experiment, the silver coated iMS nanoprobes were used todistinguish targets having a single G/C base mismatch (the signalobserved was ˜45% less than that observed for the exact-match target).

In a similar experiment, the silver coated iMS nanoprobes were embeddedin a NIPAM-hydrogel protective coating. These nanoprobes weredemonstrated to possess similar target detection capability as describedfor the iMS nanoprobes without the NIPAM-hydrogel (data not shown).

Example 9 Hybrid Sliver-Coated Gold Nanostars for Surface-Enhanced RamanScattering (SERS)

In the ongoing search for ever-brighter surface-enhanced Ramanscattering (SERS) nanoprobes, gold nanostars (AuNSs) have emerged as oneof the best geometries for producing SERS in a non-aggregated state.However, for in vivo applications, optical extinction from tissue andplasmon-matched nanoparticles can greatly attenuate the SERS intensity.Herein, the development of a new hybrid bimetallic nanostar-basedplatform that exhibits superior SERS properties is reported. In this newnanoplatform, coating AuNSs with a subtotal layer of silver (AuNS@Ag)further increased their SERS brightness by an order of magnitude whenbeing interrogated by an off-resonant excitation source.Silica-encapsulated AuNS@Ag nanoprobes were injected intra-dermally intoa rat pelt, where SERS was readily detected with higher signal-to-noisethan nanoprobes prepared from AuNS. Moreover, these off-resonanceAuNS@Ag nanoprobes did not cause any gross photothermal damage totissue, which was observed with the plasmon-matched AuNSs. ThisSERS-active hybrid nanoprobe exhibits high SERS brightness and offerspromising properties for future applications in sensing and molecularimaging.

In recent years, much effort has been devoted to the development ofnanoparticles with the brightest SERS possible. While gold and silvernanosphere colloids have long been used in SERS studies, aggregation istypically required to generate the “hot-spots” of electromagnetic fieldfor high SERS enhancement. Although this can give extremely low limitsof detection, reproducibility becomes an issue when aggregation isrelied upon. To overcome this problem, nanoparticles with intrinsichotspots, such as nanorods and AuNSs, can be employed. AuNSs exhibitsuperior SERS properties owing to their tunable plasmon, for matchingthe excitation wavelength, and multiple sharp branches, each with astrongly enhanced electromagnetic (EM) field localized at its tip.

The present experiment describes the development of nanosensor for exvivo and in vive applications, which presented several challenges. Thefirst issue was the extremely high attenuation of SERS signal whenattempting to detect the particles through tissue. The second issuefound was the efficient photothermal transduction of AuNS solution,causing unwanted localized tissue burning. It was interesting to notethat when using a commercially available SERS nanoprobes based onaggregated gold nanospheres, the signal attenuation due toself-absorption was lower and heating of the solution after laserexcitation was minimal. Such phenomena can be explained by the mismatchbetween the extinction maximum of these nanoparticles and the wavelengthof the incident laser, hence limiting photothermal transduction andself-absorption of the Raman scattered light Other reports have recentlyshown that plasmon matching is not as desirable as once thought whenperforming SERS measurements in solution. It was therefore of interestto develop highly SERS active (i.e., highest brightness factor)nanoparticles without the aforementioned disadvantages.

Two strategies were employed in this study. One was to use resonant dyesto generate resonant SERS (SERRS). The other was to modify thecomposition and plasmon band of the nanoparticles to enhance theiroptical properties through silver coating. This process allowed themonodispersity of gold nanoparticles to be preserved while takingadvantage of the superior optical properties of silver. Although silvercoating has previously been applied to various gold nanoparticles, therehave been no reports about the coating of silver on AuNSs for makingSERS nanoprobes. Herein, we describe a method to coat AuNSs withdifferent amounts of silver, resulting in an order of magnitude increasein SERS brightness. By blue-shifting the plasmon of the particles, thereis a significant decrease in the amount of self-absorption and heatgenerated when irradiating with a NIR laser. These silver-coated goldnanostars (AuNS@Ag) were used to make silica-coated SERS nanoprobes thatwere injected into rat skin ex vivo to demonstrate the utility of thislSERS platform in biological applications.

EXPERIMENTAL

Materials.

Gold(III) chloride trihydrate (HAuCl₄.3H₂O), L(+)-ascorbic acid (AA),tetraethyl orthosilicate (TEOS), trisodium citrate dihydrate, 1 Nhydrochloric acid solution (HCl), hexadecyltrimethylammonium bromide(CTAB, product H9151), Dulbecco's phosphate buffered saline (PBS),O-[2-(3-Mercaptopropionylamino)ethyl]-O′-methylpolyethylene glycol(mPEG-SH, MW 5k), IR-780 iodide, 3,3′-Diethylthiadicarbocyanine iodide(DTDC), 3,3′-Diethylthiatricarbocyanine iodide (DTTC), and1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide (HITC) werepurchased from Sigma-Aldrich (St. Louis, Mo., USA) at the highest puritygrade available. Silver nitrate (AgNO₃, 99.995%) was supplied by AlfaAesar (Ward Hill, Mass., USA). Ammonium hydroxide (NH₄OH, 29.5%),carbon-coated copper TEM grids, 1 mL disposable syringes, 27 G×1/2″needles, and 200 proof ethanol (EtOH) were obtained through VWR (Radnor,Pa.). All glassware and stir bars were thoroughly cleaned with aquaregia and dried prior to use. Ultrapure water (18 Mω

cm) was used in all preparations.

Instrumentation.

Raman spectra were recorded with a PIXIS:100BReX CCD mounted to a LS-785spectrograph (1200 g mm¹ grating), controlled by LightField software,from Princeton Instruments (Trenton, N.J.). A 785 nm diode laser wasfiber-coupled to an InPhotonics RamanProbe (Norwood, Mass.) forexcitation, with a power of 150 mW at the sample; the collection fiberof the RamanProbe was coupled to the entrance slit of the LS-785spectrograph. Absorption spectra were collected with a FLUOstar Omegaplate reader (BMG LABTECH GmbH, Germany). A FEI Tecnai G² Twintransmission electron microscope (Hillsboro, Oreg., USA) was used toacquire transmission electron microscopy (TEM) micrographs. Particlesize distributions were measured by Nanoparticle Tracking Analysis (NTA)on a NanoSight NS500 (Amesbury, UK).

Nanostar Synthesis (AuNS).

Two sizes of AuNS were synthesized as previously reported. A 12 nm goldseed solution was prepared by adding 15 mL of 1% trisodium citrate to100 mL of a boiling solution of 1 mM HAuCl₄. This solution was keptboiling for an additional 15 minutes, cooled to room temperature in anice bath, filtered through a 0.22 μm nitrocellulose membrane, and storedat 4° C. until use. To produce the larger AuNS, designated S30, 100 μLof the gold seed was added to a 10 mL solution of 0.25 mM HAuCl₄containing 10 μL of 1 N HCl, immediately followed by the simultaneousaddition of 50 μL 0.1 M AA and 100 μL 3 mM AgNO₃ under moderatestirring. The smaller AuNS, designated S5, were produced in the samemanner as above, but using 0.5 mM AgNO₃ in place of 3 mM AgNO₃. Aftersynthesis, 100 μL of 0.1 M CTAB was added to the AuNS solution and leftstirring for 5 minutes. The particles were then centrifuged at 2000 rcffor 20 minutes at 4° C., the supernatant discarded, and the particlesre-dispersed in 10 mL of 1 mM CTAB solution.

Silver Coating of Gold Nanostars (AuNS@Ag). A 1 mL aliquot of the washedAuNS solution was transferred into a 1.5 mL centrifuge tube. The samplewas briefly vortexed after each subsequent chemical addition. A smallvolume (varied between 0 and 15 μL) of 0.1 M AgNO₃ and an equivalentvolume of 0.1 M AA were added to the solution. The reduction of silverby AA was initiated by the addition of NH₄OH (same volume as above), atwhich point the color of the solution began to darken. After about 5minutes, the solution color had stabilized, indicating completion of thereaction. The various silver-coated AuNS samples were designatedaccording to the volume of AgNO₃ added (e.g., S30@Ag5 for S30 AuNScoated using 5 μL of 0.1 M AgNO₃). The silver coated AuNS were thenlabeled with dye by adding 1 μM final concentration of the desired dye(dissolved in EtOH) to the solution, allowing it to sit for 15 minutes,centrifuging at 2000 rcf for 10 minutes, discarding the supernatant, andre-dispersing in water.

Silica Coating (AuNS@Ag@SiO₂).

Silica was coated onto the labeled AuNS@Ag using an establishedprotocol. To the 1 mL sample of dye-labeled particles prepared above, 5μL of 1 mM mPEG-SH was added and allowed to react for 1 hour. Thesolution was washed once by centrifugation (2500 rcf, 10 min) and thendispersed in 900 μL of EtOH with 200 μL of water. Silica coating wasinitiated by adding 18 μL of NH₄OH followed by 5 μL of 10% TEOS in EtOHto the solution. The reaction was allowed to proceed for 12 hours, atwhich point the sample was washed twice by centrifugation at 3000 rcffor 5 minutes and re-dispersed in water.

SERS Nanoprobe Injections.

A shaved rat pelt was provided by Dr. Bruce Klitzman. Prior toinjection, 1 mL of AuNS@Ag@SiO₂ were centrifuged at 3000 rcf for 5minutes and the supernatant discarded. The particles were thenre-dispersed in 100 μL of PBS, giving a particle concentration of about1 nM. A 1 mL disposable syringe with a 27G needle was used to draw up˜50 pL of the PBS particle solution. The needle was insertedtangentially to the skin (intradermal) with the bevel facing upward and˜25 μL of the solution was injected. The rat pelt was then placed underthe focus of the RamanProbe to collect SERS spectra.

Synthesis and Characterization.

The AuNSs used in this study were prepared as described previouslyabove. To better characterize the silver coating process, two types ofAuNSs were prepared: S5, which have low branch numbers, an averageparticle size around 50 nm, and an extinction maximum at 650 nm; andS30, which have high branching, an average particle size around 70 nm,and an extinction maximum at 850 nm. After synthesis, CTAB was added asa surfactant to stabilize the particles, which were then purified bycentrifugation to remove any unreacted reagents. Nanoparticle samplesare designated as described in the experimental section.

Silver coating of the AuNSs was performed in a similar manner toprevious reports on the coating of gold nanorods with silver. In thismethod, the CTAB-stabilized AuNSs are used as seeds for the growth of asilver shell. Ascorbic acid serves as the reducing agent, with silvernitrate used as the precursor to elemental silver. After adding AA andAgNO₃ to the AuNS seed solution, NH₄OH is introduced to increase the pH,initiating the reduction of Ag⁺ to Ag⁰ by AA. An immediate color changeis observed after the pH is adjusted and the extinction maximum of thesolution begins to blue-shift from the NIR region. After about 5minutes, the color of the solution stabilizes, indicating completion ofthe silver coating reaction. The morphology of these particles aftercoating with different amounts of silver was investigated by TEM (datanot shown). It was observed that the silver deposition begins mainly onthe core of the particles, spreading outward as the amount of silver isincreased until the branches are completely covered, resulting in aquasi-spherical shape. As can be expected, the smaller S5 have theirbranches mostly covered at lower amounts of silver than the larger S30.

To further confirm the growth of silver onto the AuNS, as opposed tonucleation of isolated silver nanospheres, UVNis absorption spectroscopywas employed (data not shown). For both S5@Ag and S30@Ag, the extinctionmaximum was blue-shifted to ˜500 nm and increased in intensity withincreasing amounts of silver. No peak was observed at ˜420 nm, where theplasmon peak of silver nanospheres occurs. The blue-shifting AuNSplasmon, along with the absence of a plasmon peak at ˜420 nm areindicative of silver shell formation on the AuNS.

To fabricate the strong SERS nanoprobes with the highest brightness,several factors were taken into consideration. Resonant SERS wasemployed because it generates multiple orders of magnitude higher SERSsignal than non-resonant SERS on non-aggregated AuNS. In addition, ithas been demonstrated that when using resonant dyes, a plasmon that isblue-shifted from the excitation provides the highest signal, asself-absorption effects are minimized when the plasmon is off-resonancefrom the excitation. Previously, sodium dodecyl sulfate (SDS) was usedas a surfactant on AuNSs to aid in stabilization and dye adsorption. Itis believed that the hydrophobic bilayer formed by the SDS helps toentrap dyes near the particle surface. It has also been demonstratedthat CTAB can act in the same manner, and exhibits about two to threetimes higher signal intensity than particles stabilized with SDS. Thelonger hydrophobic chain of CTAB (16 carbons) versus SDS (12 carbons)likely provides a larger volume for trapping dye molecules.

For SERS intensity evaluation, the overall SERS brightness of thenanoparticle samples was compared in lieu of calculating theirenhancement factors, which tend to be inaccurate as a consequence ofassumptions made in their determination. Factors that would interferewith an enhancement factor calculation include: the irregular shape ofthe nanoparticles making it difficult to calculate their surface area todetermine the number of dye molecules that can bind per particle, theuse of CTAB leading to more than a monolayer of dye coverage perparticle, and self-absorption of the particles reducing the measuredRaman signal.

To investigate the effect of the various silver coatings on Ramanenhancement, AuNSs@Ag samples were labeled with a NIR resonant dye,IR-780, for surface-enhanced resonant Raman scattering (SERRS)measurements (data not shown). At its highest intensity, with S5@Ag3,the signal was enhanced 16±2 times over the S5@Ag0. For S30@Ag samples,the highest intensity was observed at S30@Ag7, which is 9±1 times higherthan the signal of S30@Ag0. It is worth noting that the maximal SERSbrightness was found on AuNS with sub-total silver coating. It wasapparent that the maximum Raman signal enhancement occurs right beforethe gold tips become completely embedded in the silver shell. Moresilver does not always lead to higher SERS response. Thus, a near-totalsilver coverage can add the benefit of silver enhancement whileretaining the hot-spots from the AuNS tips to yield the strongest SERS.The lower self-absorption from the surrounding off-resonantnanoparticles plays a significant role as well. In contrast, sphericalsilver coating with a mismatched plasmon maximum but no sharp tips had aSERS brightness that was only slightly greater than NS@Ag0.

In order to make sure that the particles were not aggregated, whichwould cause anomalously high Raman signals, the size distributions ofS30@Ag were evaluated by NTA, both before and after dye labeling (datanot shown). No significant increase in particle size was observed afterdye labeling, adding confidence that the particles remained in anon-aggregated state. The observed drop-off in Raman signal intensityafter a certain amount of silver coating further supports the claim thatparticles remain non-aggregated after dye labeling. With mostlyspherical-shaped particles found in solution after the optimum amount ofsilver coating is surpassed, any type of aggregation would result in amarked increase in Raman signal.

SERS Nanoprobe Preparation.

In order to make Raman-labeled particles suitable for bio-applications,it is necessary to encapsulate the particles in an inert material, e.g.silica. Coating the Raman nanoprobes with silica helped to keep the dyetrapped on the particle surface, making them more stable, and preventedunwanted adsorption of other molecules that may generate their own Ramansignal. SERS nanoprobes were prepared using three different carbocyaninedyes (DTDC, DTTC, and HITC) to demonstrate the potential for multiplexdetection. Thiol-PEG was used to stabilize the particles whentransferred into ethanol for silica coating by a modified StOber method.The PEG layer also acted to facilitate silica condensation onto thesurface of the particles, presumably through hydrogen bonding. Thismethod was found to be equally effective at encapsulating bothsilver-coated and bare AuNS. The measured particle size distributionshowed no obvious signs of aggregation after silica coating. FIG. 24Ashows a comparison of the Raman signal intensity from silica-coated,DTTC-labeled S30@Ag0 and S30@Ag7. It is shown that after silica coating,the order of magnitude in signal difference between the Ag0 and Ag7 S30is maintained. The TEM micrographs shown in FIGS. 24B and 24Cdemonstrate that both particle types were completely coated and in anon-aggregated state.

Intradermal Injection of SERS Nanoprobes Ex Vivo.

To show the potential of the new AuNS@Ag in biological applications, theprepared SERS nanoprobes were injected into a rat pelt for ex vivedetection. To prepare the particles for injection, the solutions wereconcentrated ten times and dispersed in sterile PBS. The particlesolutions were drawn up into 1 mL syringes with a 27G needle. About 25μL of each SERS nanoprobe was injected into the skin at differentlocations. The injection volume produced a small welt, with the particlesolutions clearly visible through the skin (data not shown). Theinjection area was then swabbed with an alcohol pad before opticalinterrogation with the RamanProbe.

A Raman signal was observed for the three AuNS@Ag SERS nanoprobes (DTTC,HITC, and DTDC), as well as the DTTC AuNS SERS nanoprobe (data notshown). The S30@Ag7-DTTC@SiO₂ nanoprobe showed a higher signal-to-noiseratio than the S30@Ag0-DTTC@SiO₂ nanoprobe, demonstrating enhancedsignal generation from the silver-coated AuNS. The difference in signalintensity from resonant (DTTC and HITC) and non-resonant (DTDC) dyes wasalso observed, with the resonant dyes providing an order of magnitudehigher signal than the non-resonant dye.

Another benefit of the AuNS@Ag SERS nanoprobes is that the extinctionmaximum no longer occurs in the region of the laser excitation. Noadverse effects on the tissue were seen for the AuNS@Ag nanoprobes afterlaser interrogation. However, a small area of burnt tissue was observedin the center of the S30@Ag0-DTTC@SiO₂ injection site after themeasurement had been performed. It has previously been shown that theAuNS are efficient photothermal transducers due to their highabsorption:scattering ratio. In this case, the power of the incidentlaser was high enough to cause burning of the tissue when non-silvercoated AuNS were used. Although matching the laser excitation to thesurface plasmon resonance of nanoparticles will generate the highestelectromagnetic field enhancement for SERS, this study demonstrates thatthis is not always desirable, as doing so can lead to unintended tissuedamage.

This report described the synthesis, characterization, and applicationof a hybrid bimetallic platform, AuNS@Ag, for SERS detection. The amountof silver coating was optimized to give the greatest SERS brightness.The morphology of the particles was assessed by TEM, while the opticalproperties were characterized with UVNis absorption spectroscopy andRaman spectroscopy. In the optimized configuration, AuNS@Ag providedover an order of magnitude of signal enhancement compared to uncoatedAuNS. Three different dye-labeled AuNS@Ag were coated with a silicashell to create SERS nanoprobes, entrapping the dye and preserving thenon-aggregated state of the particles. To demonstrate the utility ofthese particles in bio-labeling applications, ex vive detection wasperformed following intradermal injection of the SERS nanoprobes into arat pelt. Raman signal was detected from all three SERS nanoprobes, andthe measurements did not cause any noticeable damage to the skin. TheSERS nanoprobe created from non-silver-coated AuNS caused burning of thetissue after laser irradiation, due to photothermal conversion caused bythe overlap between the particles' surface plasmon resonance and thewavelength of the incident light.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which thepresent disclosure pertains. These patents and publications are hereinincorporated by reference in their entiretly to the same extent as ifeach individual publication was specifically and individually indicatedto be incorporated by reference.

One skilled in the art will readily appreciate that the present presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods described herein are presentlyrepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the present disclosure as defined bythe scope of the claims.

1-22. (canceled)
 23. An inverse molecular sentinel nanoprobe fordetecting a nucleic acid target, comprising a metal nanoparticle, anoligonucleotide molecule attached at a first end to the nanoparticle,the oligonucleotide molecule comprising a stem-L sequence and a stem-Rsequence capable of hybridizing to form a hairpin structure, and aplaceholder binding sequence between the stem-L sequence and the stem-Rsequence, a separate placeholder strand, which is at least partiallycomplementary to the placeholder binding sequence of the oligonucleotidemolecule, wherein the placeholder strand is hybridized to theplaceholder binding sequence in the absence of the nucleic acid targetsuch that the oligonucleotide molecule is in a non-hairpin configurationwhen the placeholder strand is hybridized thereto; and an optical labelattached to a second end of the oligonucleotide molecule, wherein thesecond end is opposite the first end of the oligonucleotide molecule,and wherein, in the presence of the nucleic acid target, a hairpinstructure forms in the oligonucleotide molecule as a result of theplaceholder strand competitively binding to the nucleic acid target, andwherein the formation of the hairpin structure in the oligonucleotidemolecule places the optical label in the proximity of the metalnanoparticle, thereby resulting in an increase in intensity of signalemitted from the optical label relative to the intensity emitted in theabsence of the nucleic acid target.
 24. The nanoprobe of claim 23,wherein the optical label comprises a Raman dye,3,3′-Diethylthiadicarbocyanine iodide (DTDC),3,3′-diethylthiatricarbocyanineiodide (DTTC),1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide (HITC), CY3 dye,CY3.5 dye, CY5.5 dye, CY7 dye, CY7.5 dye, a positively-chargedhydrophobic near infrared (NIR) dye, IR-780, IR-792, IR-797, IR-813,methylene blue hydrate (MB), 4-mercaptobenzoic acid (4-MBA),5,5′-dithiobis-2-nitrobenzoic acid (DTNB), 4-aminothiophenol (4ATP),fluorescein, fluorescein isothiocyanate (FITC), thionine dyes,rhodamine-based dye, crystal violet, a fluorescence label, or absorbancelabel.
 25. The nanoprobe of claim 23, wherein the nucleic acid targetcomprises a DNA, an RNA, a microRNA, a mRNA, or a single polynucleotidepolymorphism (SNP).
 26. The nanoprobe of claim 23, wherein theplaceholder strand is an siRNA or an anti-microRNA.
 27. The nanoprobe ofclaim 23, wherein the metal nanoparticle comprises silver nanoparticles,gold nanoparticles, silver nanostars, gold nanostars, silver-coated goldnanostars, bimetallic nanoparticles, multi-metallic nanoparticles,dielectric nanoparticle cores covered with metal nanoshells, ormulti-nanoparticle structures.
 28. The nanoprobe of claim 23, whereinthe metal nanoparticle comprises a gold nanostar.
 29. The nanoprobe ofclaim 23, wherein the metal nanoparticle is embedded in a hollow silicashell.
 30. An inverse molecular sentinel nanoprobe for detecting anucleic acid target, comprising a metal nanoparticle, an oligonucleotidemolecule attached at a first end to the nanoparticle, theoligonucleotide molecule comprising a stem-L sequence and a stem-Rsequence capable of hybridizing to form a hairpin structure, and aspacer and a placeholder binding sequence between the stem-L sequenceand the stem-R sequence, wherein the placeholder binding sequence andthe spacer do not overlap and the placeholder binding sequenceoptionally overlaps the stem-R sequence; a separate placeholder strand,which is at least partially complementary to the placeholder bindingsequence of the oligonucleotide molecule, wherein the placeholder strandis hybridized to the placeholder binding sequence in the absence of thenucleic acid target such that the oligonucleotide molecule is in anon-hairpin configuration when the placeholder strand is hybridizedthereto; and an optical label attached to a second end of theoligonucleotide molecule, wherein the second end is opposite the firstend of the oligonucleotide molecule, and wherein, in the presence of thenucleic acid target, a hairpin structure forms in the oligonucleotidemolecule as a result of the placeholder strand competitively binding tothe nucleic acid target, and wherein the formation of the hairpinstructure in the oligonucleotide molecule places the optical label inthe proximity of the metal nanoparticle, thereby resulting in anincrease in intensity of signal emitted from the optical label relativeto the intensity emitted in the absence of the nucleic acid target. 31.The nanoprobe of claim 30, wherein the optical label comprises a Ramandye, 3,3′-Diethylthiadicarbocyanine iodide (DTDC),3,3′-diethylthiatricarbocyanineiodide (DTTC),1,1′,3,3,3′,3′-Hexamethylindotricarbocyanine iodide (HITC), CY3 dye,CY3.5 dye, CY5.5 dye, CY7 dye, CY7.5 dye, a positively-chargedhydrophobic near infrared (NIR) dye, IR-780, IR-792, IR-797, IR-813,methylene blue hydrate (MB), 4-mercaptobenzoic acid (4-MBA),5,5′-dithiobis-2-nitrobenzoic acid (DTNB), 4-aminothiophenol (4ATP),fluorescein, fluorescein isothiocyanate (FITC), thionine dyes,rhodamine-based dye, crystal violet, a fluorescence label, or absorbancelabel.
 32. The nanoprobe of claim 30, wherein the nucleic acid targetcomprises a DNA, an RNA, a microRNA, a mRNA, or a single polynucleotidepolymorphism (SNP).
 33. The nanoprobe of claim 30, wherein theplaceholder strand is an siRNA or an anti-microRNA.
 34. The nanoprobe ofclaim 30, wherein the metal nanoparticle comprises silver nanoparticles,gold nanoparticles, silver nanostars, gold nanostars, silver-coated goldnanostars, bimetallic nanoparticles, multi-metallic nanoparticles,dielectric nanoparticle cores covered with metal nanoshells, ormulti-nanoparticle structures.
 35. The nanoprobe of claim 30, whereinthe metal nanoparticle comprises a gold nanostar.
 36. The nanoprobe ofclaim 30, wherein the metal nanoparticle is embedded in a hollow silicashell.